U.S. patent application number 16/309453 was filed with the patent office on 2019-08-22 for fish allergy antigen.
The applicant listed for this patent is HOYU CO., LTD.. Invention is credited to Kayoko Matsunaga, Naoshi Shimojo, Akiko Yagami.
Application Number | 20190256566 16/309453 |
Document ID | / |
Family ID | 60663992 |
Filed Date | 2019-08-22 |
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United States Patent
Application |
20190256566 |
Kind Code |
A1 |
Matsunaga; Kayoko ; et
al. |
August 22, 2019 |
FISH ALLERGY ANTIGEN
Abstract
The present invention provides novel antigens of an allergy to
fish, methods and kits for diagnosing an allergy to fish, a
pharmaceutical composition comprising the antigen, fish or fish
eggs in which the antigen is eliminated, a processed product of the
fish or the fish eggs or fish which lay the fish eggs or have
hatched from the eggs, and a tester for determining the presence or
absence of a fish antigen in an object of interest.
Inventors: |
Matsunaga; Kayoko; (Aichi,
JP) ; Yagami; Akiko; (Aichi, JP) ; Shimojo;
Naoshi; (Aichi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HOYU CO., LTD. |
Aichi |
|
JP |
|
|
Family ID: |
60663992 |
Appl. No.: |
16/309453 |
Filed: |
April 28, 2017 |
PCT Filed: |
April 28, 2017 |
PCT NO: |
PCT/JP2017/017018 |
371 Date: |
December 13, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 2800/24 20130101; C12N 15/09 20130101; C12Q 1/48 20130101;
A23L 17/30 20160801; A23V 2002/00 20130101; A23L 17/00 20160801;
C07K 14/47 20130101; A61K 39/00 20130101; G01N 33/68 20130101; A61P
37/08 20180101; C07K 14/461 20130101; G01N 33/53 20130101 |
International
Class: |
C07K 14/46 20060101
C07K014/46; A23L 17/00 20060101 A23L017/00; A23L 17/30 20060101
A23L017/30; G01N 33/68 20060101 G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 16, 2016 |
JP |
2016-119919 |
Claims
1. A kit for diagnosing a fish allergy, the kit comprising, as an
antigen, at least one protein defined in any one of the following
(1) to (9): (1) (1A) Alpha-actinin-3 or a variant thereof, which is
a protein defined below in any of (1A-a) to (1A-e) which is an
antigen for the fish allergy: (1A-a) a protein comprising an amino
acid sequence with deletion, substitution, insertion or addition of
one or several amino acids in SEQ ID NO: 2; (1A-b) a protein
comprising an amino acid sequence having at least 70% identity to
the amino acid sequence of SEQ ID NO: 2; (1A-c) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
with deletion, substitution, insertion or addition of one or
several nucleotides in SEQ ID NO: 1; (1A-d) a protein comprising an
amino acid sequence encoded by a nucleotide sequence having at
least 70% identity to the nucleotide sequence of SEQ ID NO: 1; or
(1A-e) a protein comprising an amino acid sequence encoded by a
nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 1; or (1B) a protein comprising
an amino acid sequence of SEQ ID NO: 2 or 3; (2) (2A) EEF1A2
binding protein-like or a variant thereof, which is a protein
defined below in any of (2A-a) to (2A-e) which is an antigen for
the fish allergy: (2A-a) a protein comprising an amino acid
sequence with deletion, substitution, insertion or addition of one
or several amino acids in SEQ ID NO: 5; (2A-b) a protein comprising
an amino acid sequence having at least 70% identity to the amino
acid sequence of SEQ ID NO: 5; (2A-c) a protein comprising an amino
acid sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 4; (2A-d) a protein comprising an amino acid sequence
encoded by a nucleotide sequence having at least 70% identity to
the nucleotide sequence of SEQ ID NO: 4; or (2A-e) a protein
comprising an amino acid sequence encoded by a nucleic acid that
hybridizes under stringent conditions with a nucleic acid having a
nucleotide sequence complementary to the nucleotide sequence of SEQ
ID NO: 4; or (2B) a protein comprising at least one amino acid
sequence selected from the group consisting of SEQ ID NOs: 5-8; (3)
(3A) Alpha-1,4-glucan phosphorylase or a variant thereof, which is
a protein defined below in any of (3A-a) to (3A-e) which is an
antigen for the fish allergy: (3A-a) a protein comprising an amino
acid sequence with deletion, substitution, insertion or addition of
one or several amino acids in SEQ ID NO: 10; (3A-b) a protein
comprising an amino acid sequence having at least 70% identity to
the amino acid sequence of SEQ ID NO: 10; (3A-c) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
with deletion, substitution, insertion or addition of one or
several nucleotides in SEQ ID NO: 9; (3A-d) a protein comprising an
amino acid sequence encoded by a nucleotide sequence having at
least 70% identity to the nucleotide sequence of SEQ ID NO: 9; or
(3A-e) a protein comprising an amino acid sequence encoded by a
nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 9; or (3B) a protein comprising
at least one amino acid sequence selected from the group consisting
of SEQ ID NOs: 10-16; (4) (4A) Elongation factor 2 or a variant
thereof, which is a protein defined below in any of (4A-a) to
(4A-e) which is an antigen for the fish allergy: (4A-a) a protein
comprising an amino acid sequence with deletion, substitution,
insertion or addition of one or several amino acids in SEQ ID NO:
19; (4A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 19;
(4A-c) a protein comprising an amino acid sequence encoded by a
nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 18; (4A-d) a
protein comprising an amino acid sequence encoded by a nucleotide
sequence having at least 70% identity to the nucleotide sequence of
SEQ ID NO: 18; or (4A-e) a protein comprising an amino acid
sequence encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 18; or (4B)
a protein comprising at least one amino acid sequence selected from
the group consisting of SEQ ID NOs: 19-24; (5) (5A) Heat shock
cognate 70 kDa protein or a variant thereof, which is a protein
defined below in any of (5A-a) to (5A-e) which is an antigen for
the fish allergy: (5A-a) a protein comprising an amino acid
sequence with deletion, substitution, insertion or addition of one
or several amino acids in SEQ ID NO: 26; (5A-b) a protein
comprising an amino acid sequence having at least 70% identity to
the amino acid sequence of SEQ ID NO: 26; (5A-c) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
with deletion, substitution, insertion or addition of one or
several nucleotides in SEQ ID NO: 25; (5A-d) a protein comprising
an amino acid sequence encoded by a nucleotide sequence having at
least 70% identity to the nucleotide sequence of SEQ ID NO: 25; or
(5A-e) a protein comprising an amino acid sequence encoded by a
nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 25; or (5B) a protein comprising
at least one amino acid sequence selected from the group consisting
of SEQ ID NOs: 26-31; (6) (6A) Serotransferrin or a variant
thereof, which is a protein defined below in any of (6A-a) to
(6A-e) which is an antigen for the fish allergy: (6A-a) a protein
comprising an amino acid sequence with deletion, substitution,
insertion or addition of one or several amino acids in SEQ ID NO:
33; (6A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 33;
(6A-c) a protein comprising an amino acid sequence encoded by a
nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 32; (6A-d) a
protein comprising an amino acid sequence encoded by a nucleotide
sequence having at least 70% identity to the nucleotide sequence of
SEQ ID NO: 32; or (6A-e) a protein comprising an amino acid
sequence encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 32; or (6B)
a protein comprising at least one amino acid sequence selected from
the group consisting of SEQ ID NOs: 33-41; (7) (7A) Myosin binding
protein H-like or a variant thereof, which is a protein defined
below in any of (7A-a) to (7A-e) which is an antigen for the fish
allergy: (7A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 43; (7A-b) a protein comprising an amino
acid sequence having at least 70% identity to the amino acid
sequence of SEQ ID NO: 43; (7A-c) a protein comprising an amino
acid sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 42; (7A-d) a protein comprising an amino acid
sequence encoded by a nucleotide sequence having at least 70%
identity to the nucleotide sequence of SEQ ID NO: 42; or (7A-e) a
protein comprising an amino acid sequence encoded by a nucleic acid
that hybridizes under stringent conditions with a nucleic acid
having a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 42; or (7B) a protein comprising at least
one amino acid sequence selected from the group consisting of SEQ
ID NOs: 43-54, (8) (8A) Desmin (Fragment) or a variant thereof,
which is a protein defined below in any of (8A-a) to (8A-e) which
is an antigen for the fish allergy: (8A-a) a protein comprising an
amino acid sequence with deletion, substitution, insertion or
addition of one or several amino acids in SEQ ID NO: 56; (8A-b) a
protein comprising an amino acid sequence having at least 70%
identity to the amino acid sequence of SEQ ID NO: 56; (8A-c) a
protein comprising an amino acid sequence encoded by a nucleotide
sequence with deletion, substitution, insertion or addition of one
or several nucleotides in SEQ ID NO: 55; (8A-d) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
having at least 70% identity to the nucleotide sequence of SEQ ID
NO: 55; or (8A-e) a protein comprising an amino acid sequence
encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 55; or (8B)
a protein comprising at least one amino acid sequence selected from
the group consisting of SEQ ID NOs: 56-59; (9) (9A) Capping protein
(Actin filament) muscle Z-line beta or a variant thereof, which is
a protein defined below in any of (9A-a) to (9A-e) which is an
antigen for the fish allergy: (9A-a) a protein comprising an amino
acid sequence with deletion, substitution, insertion or addition of
one or several amino acids in SEQ ID NO: 61; (9A-b) a protein
comprising an amino acid sequence having at least 70% identity to
the amino acid sequence of SEQ ID NO: 61; (9A-c) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
with deletion, substitution, insertion or addition of one or
several nucleotides in SEQ ID NO: 60; (9A-d) a protein comprising
an amino acid sequence encoded by a nucleotide sequence having at
least 70% identity to the nucleotide sequence of SEQ ID NO: 60; or
(9A-e) a protein comprising an amino acid sequence encoded by a
nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 60; or (9B) a protein comprising
at least one amino acid sequence selected from the group consisting
of SEQ ID NOs: 61-68.
2. A kit for diagnosing a fish allergy, the kit comprising, as an
antigen, at least one protein defined in any one of the following
(10) to (14): (10) (10A) Myosin heavy chain, fast skeletal
muscle-like or a variant thereof, which is a protein defined below
in any of (10A-a) to (10A-e) which is an antigen for the fish
allergy: (10A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 70; (10A-b) a protein comprising an amino
acid sequence having at least 70% identity to the amino acid
sequence of SEQ ID NO: 70; (10A-c) a protein comprising an amino
acid sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 69; (10A-d) a protein comprising an amino acid
sequence encoded by a nucleotide sequence having at least 70%
identity to the nucleotide sequence of SEQ ID NO: 69; or (10A-e) a
protein comprising an amino acid sequence encoded by a nucleic acid
that hybridizes under stringent conditions with a nucleic acid
having a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 69; or (10B) a protein comprising at least
one amino acid sequence selected from the group consisting of SEQ
ID NOs: 70-108; (11) (11A) Glycogen phosphorylase, muscle form-like
or a variant thereof, which is a protein defined below in any of
(11A-a) to (11A-e) which is an antigen for the fish allergy:
(11A-a) a protein comprising an amino acid sequence with deletion,
substitution, insertion or addition of one or several amino acids
in SEQ ID NO: 110; (11A-b) a protein comprising an amino acid
sequence having at least 70% identity to the amino acid sequence of
SEQ ID NO: 110; (11A-c) a protein comprising an amino acid sequence
encoded by a nucleotide sequence with deletion, substitution,
insertion or addition of one or several nucleotides in SEQ ID NO:
109; (11A-d) a protein comprising an amino acid sequence encoded by
a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 109; or (11A-e) a protein
comprising an amino acid sequence encoded by a nucleic acid that
hybridizes under stringent conditions with a nucleic acid having a
nucleotide sequence complementary to the nucleotide sequence of SEQ
ID NO: 109; or (11B) a protein comprising at least one amino acid
sequence selected from the group consisting of SEQ ID NOs: 110-119;
(12) (12A) Myosin-binding protein C, fast-type-like or a variant
thereof, which is a protein defined below in any of (12A-a) to
(12A-e) which is an antigen for the fish allergy: (12A-a) a protein
comprising an amino acid sequence with deletion, substitution,
insertion or addition of one or several amino acids in SEQ ID NO:
121; (12A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 121;
(12A-c) a protein comprising an amino acid sequence encoded by a
nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 120; (12A-d) a
protein comprising an amino acid sequence encoded by a nucleotide
sequence having at least 70% identity to the nucleotide sequence of
SEQ ID NO: 120; or (12A-e) a protein comprising an amino acid
sequence encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 120; or
(12B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 121-136; (13)
(13A) ATP synthase subunit beta, mitochondrial or a variant
thereof, which is a protein defined below in any of (13A-a) to
(13A-e) which is an antigen for the fish allergy: (13A-a) a protein
comprising an amino acid sequence with deletion, substitution,
insertion or addition of one or several amino acids in SEQ ID NO:
138; (13A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 138;
(13A-c) a protein comprising an amino acid sequence encoded by a
nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 137; (13A-d) a
protein comprising an amino acid sequence encoded by a nucleotide
sequence having at least 70% identity to the nucleotide sequence of
SEQ ID NO: 137; or (13A-e) a protein comprising an amino acid
sequence encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 137; or
(13B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 138-142; (14)
(14A) L-lactate dehydrogenase A chain-like or a variant thereof,
which is a protein defined below in any of (14A-a) to (14A-e) which
is an antigen for the fish allergy: (14A-a) a protein comprising an
amino acid sequence with deletion, substitution, insertion or
addition of one or several amino acids in SEQ ID NO: 144; (14A-b) a
protein comprising an amino acid sequence having at least 70%
identity to the amino acid sequence of SEQ ID NO: 144; (14A-c) a
protein comprising an amino acid sequence encoded by a nucleotide
sequence with deletion, substitution, insertion or addition of one
or several nucleotides in SEQ ID NO: 143; (14A-d) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
having at least 70% identity to the nucleotide sequence of SEQ ID
NO: 143; or (14A-e) a protein comprising an amino acid sequence
encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 143; or
(14B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 144-149.
3. A composition for diagnosing a fish allergy, the composition
comprising, as an antigen, at least one of proteins as defined in
any of (1) to (9) of claim 1.
4. A method for providing an indicator for diagnosing a fish
allergy in a subject, the method comprising the steps of: (i)
contacting a sample obtained from the subject with an antigen,
wherein the sample is a solution comprising an IgE antibody; (ii)
detecting binding between the IgE antibody present in the sample
obtained from the subject and the antigen; and (iii) when the
binding between the IgE antibody in the subject and the antigen is
detected, an indicator of the fact that the subject has a fish
allergy is provided; wherein the antigen is at least one of
proteins as defined in any of (1) to (9) of claim 1.
5. A pharmaceutical composition comprising at least one of proteins
as defined in any of (1) to (9) of claim 1.
6. The pharmaceutical composition according to claim 5 for the
treatment of an allergy to a fish.
7. A fish or a processed product of fish in which an antigen is
eliminated or reduced, wherein the antigen is at least one of
proteins as defined in any of (1) to (9) of claim 1.
8. A tester for determining the presence or absence of a fish
antigen in an object of interest, the tester comprising an antibody
that binds to at least one of proteins as defined in any of (1) to
(9) of claim 1.
9. A tester for determining the presence or absence of an antigen
causing an allergy to fish in an object of interest, the tester
comprising a primer having a nucleotide sequence complementary to
at least one part of one of the nucleotide sequences set forth in
SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, or
143.
10. A fish-derived antigen, which is at least one of proteins as
defined in any of (1) to (9) of claim 1 and causes an allergy to a
fish.
11. A composition for diagnosing a fish allergy, the composition
comprising, as an antigen, at least one of proteins as defined in
any of (10) to (14) of claim 2.
12. A method for providing an indicator for diagnosing a fish
allergy in a subject, the method comprising the steps of: (i)
contacting a sample obtained from the subject with an antigen,
wherein the sample is a solution comprising an IgE antibody; (ii)
detecting binding between the IgE antibody present in the sample
obtained from the subject and the antigen; and (iii) when the
binding between the IgE antibody in the subject and the antigen is
detected, an indicator of the fact that the subject has a fish
allergy is provided; wherein the antigen is at least one of
proteins as defined in any of (10) to (14) of claim 2.
13. A pharmaceutical composition comprising at least one of
proteins as defined in any of (10) to (14) of claim 2.
14. The pharmaceutical composition according to claim 13 for the
treatment of an allergy to a fish.
15. A fish or a processed product of fish in which an antigen is
eliminated or reduced, wherein the antigen is at least one of
proteins as defined in any of (10) to (14) of claim 2.
16. A tester for determining the presence or absence of a fish
antigen in an object of interest, the tester comprising an antibody
that binds to at least one of proteins as defined in any of (10) to
(14) of claim 2.
17. A fish-derived antigen, which is at least one of proteins as
defined in any of (10) to (14) of claim 2 and causes an allergy to
a fish.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel antigen of an
allergy to fish. The present invention also relates to a kit, a
composition, and a method for diagnosing allergy to fish. The
present invention also relates to a pharmaceutical composition
comprising the antigen and fish or fish eggs in which the antigen
is eliminated or reduced, a processed product of the fish or the
fish eggs or fish which lay the fish eggs or have hatched from the
eggs. The present invention further relates to a tester for
determining the presence or absence of a fish antigen in an object
of interest.
BACKGROUND ART
[0002] In blood and tissues of allergic patients, IgE antibodies
specific to particular antigens are produced. Physiological
consequences caused by interaction between such IgE antibodies and
such particular antigens elicit allergic reactions.
[0003] In the process of production of conventional allergy testing
agents, antigen reagents are commonly prepared simply by grinding a
candidate allergenic food, food material or the like (Patent
Literature 1). Therefore, numerous proteins were contained in a
reagent and there the content of each protein was very little. For
this reason, the only case where conventional allergy tests have
permitted detection of a positive allergic reaction is when in a
conventional antigen reagent containing many proteins, a particular
antigen protein which causes the allergic reaction (allergen
component) is present in an amount exceeding a threshold that
allows determination of a positive reaction for binding to an IgE
antibody. However, no determination of a positive reaction was
possible and diagnosis efficiency was not sufficiently high when
using a conventional allergy testing agent in patients possessing
an IgE antibody binding to an allergen component present in small
amounts in an allergen such as food, food material or the like.
[0004] The severity and symptoms of an allergic reaction do not
necessarily correlate with the content of an allergen component.
Even when a patient's IgE antibody reacts with an allergen
component present in trace amounts in a candidate allergic food and
food material, the allergic reaction may develop allergic symptoms
or may affect the severity of those symptoms.
[0005] An attempt to increase the diagnostic efficiency is being
made by examining IgE antibodies to each protein composing food and
food material to distinguish sensitization that directly
contributes to a diagnosis from sensitization based on
cross-antigenicity by a pan-allergen or the like. The allergens set
forth in Table below are currently known as fish allergens (Non
Patent Literature 1).
[0006] However, while it is necessary to exhaustively identify
allergen components in candidate allergic foods and food materials
in order to enhance the reliability of allergy tests, the patient
detection rate by the measurement of such allergen components is
far from sufficient. Identification of novel allergens in fish is
very important not only for increasing the precision of diagnosis,
but also for determining targets of therapeutic agents and low
allergenic foods and low allergenic food materials.
[0007] Meanwhile, in the field of protein separation and
purification, various efforts have conventionally been made to
develop methods for separating and purifying a protein or nucleic
acid of interest from cell extracts or the like. Such methods may
well be exemplified by dialysis based on salt concentration, and
centrifugal separation.
[0008] Other efforts have been made to develop many purification
methods based on electric charges of protein or nucleic acid
residues or on the difference in molecular weight. Electric
charge-based purification methods can be exemplified by column
chromatography using ion exchange resins, and isoelectric focusing.
Purifications based on molecular weight difference can be
exemplified by centrifugal separation, molecular-sieve column
chromatography, and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide
gel electrophoresis).
[0009] In recent years, a method for separating and purifying many
different proteins from a small amount of sample has been used,
which is more specifically a two-dimensional electrophoresis
consisting of isoelectric focusing in the first dimension, followed
by SDS-PAGE in the second dimension. The present applicant has
conventionally developed some 2D electrophoresis methods with high
separation ability (Patent Literature 2-5).
CITATION LIST
Patent Literature
[0010] PTL1: Japanese Patent Application Publication No. JP
2002-286716
[0011] PTL2: Japanese Patent Application Publication No. JP
2011-33544
[0012] PTL3: Japanese Patent Application Publication No. JP
2011-33546
[0013] PTL4: Japanese Patent Application Publication No. JP
2011-33547
[0014] PTL5: Japanese Patent Application Publication No. JP
2011-33548
Non-Patent Literature
[0015] NPTL1: Allergen Nomenclature, WHO/IUIS Allergen Nomenclature
Sub-Committee, [searched on Jun. 9, 2016], internet<URL:
http://www.allergen.org/search.php?allergenname=&allergensource=&TaxSourc-
e=Animalia+Chordata&TaxOrder=&foodallerg=1&bioname=>
SUMMARY OF INVENTION
Technical Problem
[0016] The present invention provides novel antigens of an allergy
to fish. The present invention also provides methods and kits for
diagnosing allergy to fish. The present invention also provides
pharmaceutical compositions comprising such an antigen and fish or
fish eggs in which such an antigen is eliminated or reduced, a
processed product of the fish or the fish eggs or fish which lay
the fish eggs or have hatched from the eggs.
Solution to Problem
[0017] In order to solve the aforementioned problems, the present
inventors had made intensive studies to identify causative antigens
of an allergy to fish. As a result, the inventors succeeded in
identifying novel antigens to which an IgE antibody in the serum of
a fish-allergic patient specifically binds. The present invention
has been completed based on this finding.
[0018] Thus, in one embodiment, the present invention can be as
defined below.
[0019] [1] A kit for diagnosing a fish allergy, the kit comprising,
as an antigen, at least one protein defined in any one of the
following (1) to (9): [0020] (1) (1A) Alpha-actinin-3 or a variant
thereof, which is a protein defined below in any of (1A-a) to
(1A-e) which is an antigen for the fish allergy:
[0021] (1A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 2;
[0022] (1A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 2;
[0023] (1A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 1;
[0024] (1A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 1; or (1A-e) a protein comprising
an amino acid sequence encoded by a nucleic acid that hybridizes
under stringent conditions with a nucleic acid having a nucleotide
sequence complementary to the nucleotide sequence of SEQ ID NO: 1;
or (1B) a protein comprising an amino acid sequence of SEQ ID NO: 2
or 3; [0025] (2) (2A) EEF1A2 binding protein-like or a variant
thereof, which is a protein defined below in any of (2A-a) to
(2A-e) which is an antigen for the fish allergy:
[0026] (2A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 5;
[0027] (2A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 5;
[0028] (2A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 4;
[0029] (2A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 4; or
[0030] (2A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 4; or
[0031] (2B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 5-8; [0032] (3)
(3A) Alpha-1,4-glucan phosphorylase or a variant thereof, which is
a protein defined below in any of (3A-a) to (3A-e) which is an
antigen for the fish allergy:
[0033] (3A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 10;
[0034] (3A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 10;
[0035] (3A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 9;
[0036] (3A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 9; or
[0037] (3A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 9; or
[0038] (3B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 10-16; [0039] (4)
(4A) Elongation factor 2 or a variant thereof, which is a protein
defined below in any of (4A-a) to (4A-e) which is an antigen for
the fish allergy:
[0040] (4A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 19;
[0041] (4A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 19;
[0042] (4A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 18;
[0043] (4A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 18; or
[0044] (4A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 18; or
[0045] (4B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 19-24; [0046] (5)
(5A) Heat shock cognate 70 kDa protein or a variant thereof, which
is a protein defined below in any of (5A-a) to (5A-e) which is an
antigen for the fish allergy:
[0047] (5A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 26;
[0048] (5A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 26;
[0049] (5A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 25;
[0050] (5A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 25; or
[0051] (5A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 25; or
[0052] (5B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 26-31; [0053] (6)
(6A) Serotransferrin or a variant thereof, which is a protein
defined below in any of (6A-a) to (6A-e) which is an antigen for
the fish allergy:
[0054] (6A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 33;
[0055] (6A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 33;
[0056] (6A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 32;
[0057] (6A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 32; or
[0058] (6A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 32; or
[0059] (6B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 33-41; [0060] (7)
(7A) Myosin binding protein H-like or a variant thereof, which is a
protein defined below in any of (7A-a) to (7A-e) which is an
antigen for the fish allergy:
[0061] (7A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 43;
[0062] (7A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 43;
[0063] (7A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 42;
[0064] (7A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 42; or
[0065] (7A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 42; or
[0066] (7B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 43-54, [0067] (8)
(8A) Desmin (Fragment) or a variant thereof, which is a protein
defined below in any of (8A-a) to (8A-e) which is an antigen for
the fish allergy:
[0068] (8A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 56;
[0069] (8A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 56;
[0070] (8A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 55;
[0071] (8A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 55; or
[0072] (8A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 55; or
[0073] (8B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 56-59; [0074] (9)
(9A) Capping protein (Actin filament) muscle Z-line beta or a
variant thereof, which is a protein defined below in any of (9A-a)
to (9A-e) which is an antigen for the fish allergy:
[0075] (9A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 61;
[0076] (9A-b) a protein comprising an amino acid sequence having at
least 70% identity to the amino acid sequence of SEQ ID NO: 61;
[0077] (9A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 60;
[0078] (9A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 60; or
[0079] (9A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 60; or
[0080] (9B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 61-68.
[0081] [2] A composition for diagnosing a fish allergy, the
composition comprising, as an antigen, at least one of proteins as
defined above in any of (1) to (9) of [1].
[0082] [3] A method for providing an indicator for diagnosing a
fish allergy in a subject, the method comprising the steps of:
[0083] (i) contacting a sample obtained from the subject with an
antigen, wherein the sample is a solution comprising an IgE
antibody; [0084] (ii) detecting binding between an IgE antibody
present in the sample from the subject and the antigen; and [0085]
(iii) when the binding between the IgE antibody in the subject and
the antigen is detected, an indicator of the fact that the subject
has a fish allergy is provided; [0086] wherein the antigen is at
least one of proteins as defined above in any of (1) to (9) of
[1].
[0087] [4] A pharmaceutical composition comprising at least one of
proteins as defined above in any of (1) to (9) of [1].
[0088] [5] The pharmaceutical composition as set forth in [4] for
the treatment of a fish allergy.
[0089] [6] A fish or a processed product of fish, in which an
antigen is eliminated or reduced, wherein the antigen is at least
one of proteins as defined above in any of (1) to (9) of [1].
[0090] [7] A tester for determining the presence or absence of a
fish antigen in an object of interest, the tester comprising an
antibody that binds to at least one of proteins as defined above in
any of (1) to (9) of [1].
[0091] [8] A tester for determining the presence or absence of an
antigen causing an allergy to fish in an object of interest, the
tester comprising a primer having a nucleotide sequence
complementary to at least one part of one of the nucleotide
sequences set forth in SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, or
60.
[0092] [9] A kit for diagnosing a fish allergy, the kit comprising,
as an antigen, at least one protein defined in any one of the
following (10) to (14): [0093] (10) (10A) Myosin heavy chain, fast
skeletal muscle-like or a variant thereof, which is a protein
defined below in any of (10A-a) to (10A-e) which is an antigen for
the fish allergy:
[0094] (10A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 70;
[0095] (10A-b) a protein comprising an amino acid sequence having
at least 70% identity to the amino acid sequence of SEQ ID NO:
70;
[0096] (10A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 69;
[0097] (10A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 69; or
[0098] (10A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 69; or
[0099] (10B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 70-108; [0100]
(11) (11A) Glycogen phosphorylase, muscle form-like or a variant
thereof, which is a protein defined below in any of (11A-a) to
(11A-e) which is an antigen for the fish allergy:
[0101] (11A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 110;
[0102] (11A-b) a protein comprising an amino acid sequence having
at least 70% identity to the amino acid sequence of SEQ ID NO:
110;
[0103] (11A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 109;
[0104] (11A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 109; or
[0105] (11A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 109; or
[0106] (11B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 110-119; [0107]
(12) (12A) Myosin-binding protein C, fast-type-like or a variant
thereof, which is a protein defined below in any of (12A-a) to
(12A-e) which is an antigen for the fish allergy:
[0108] (12A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 121;
[0109] (12A-b) a protein comprising an amino acid sequence having
at least 70% identity to the amino acid sequence of SEQ ID NO:
121;
[0110] (12A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 120;
[0111] (12A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 120; or
[0112] (12A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 120; or
[0113] (12B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 121-136; [0114]
(13) (13A) ATP synthase subunit beta, mitochondrial or a variant
thereof, which is a protein defined below in any of (13A-a) to
(13A-e) which is an antigen for the fish allergy:
[0115] (13A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 138;
[0116] (13A-b) a protein comprising an amino acid sequence having
at least 70% identity to the amino acid sequence of SEQ ID NO:
138;
[0117] (13A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 137;
[0118] (13A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 137; or
[0119] (13A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 137; or
[0120] (13B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 138-142; [0121]
(14) (14A) L-lactate dehydrogenase A chain-like or a variant
thereof, which is a protein defined below in any of (14A-a) to
(l4A-e) which is an antigen for the fish allergy:
[0122] (14A-a) a protein comprising an amino acid sequence with
deletion, substitution, insertion or addition of one or several
amino acids in SEQ ID NO: 144;
[0123] (14A-b) a protein comprising an amino acid sequence having
at least 70% identity to the amino acid sequence of SEQ ID NO:
144;
[0124] (14A-c) a protein comprising an amino acid sequence encoded
by a nucleotide sequence with deletion, substitution, insertion or
addition of one or several nucleotides in SEQ ID NO: 143;
[0125] (14A-d) a protein comprising an amino acid sequence encoded
by a nucleotide sequence having at least 70% identity to the
nucleotide sequence of SEQ ID NO: 143; or
[0126] (14A-e) a protein comprising an amino acid sequence encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid having a nucleotide sequence complementary to the
nucleotide sequence of SEQ ID NO: 143; or
[0127] (14B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 144-149.
[0128] [10] A composition for diagnosing a fish allergy, the
composition comprising, as an antigen, at least one of proteins as
defined above in any of (10) to (14) of [9].
[0129] [11] A method for providing an indicator for diagnosing a
fish allergy in a subject, the method comprising the steps of:
[0130] (i) contacting a sample obtained from the subject with an
antigen, wherein the sample is a solution comprising an IgE
antibody; [0131] (ii) detecting binding between an IgE antibody
present in the sample from the subject and the antigen; and [0132]
(iii) when the binding between the IgE antibody in the subject and
the antigen is detected, an indicator of the fact that the subject
has a fish allergy is provided; [0133] wherein the antigen is at
least one of proteins as defined above in any of (10) to (14) of
[9].
[0134] [12] A pharmaceutical composition comprising at least one of
proteins as defined above in any of (10) to (14) of [9].
[0135] [13] The pharmaceutical composition as set forth in [12] for
the treatment of a fish allergy.
[0136] [14] A fish or a processed product of fish, in which an
antigen is eliminated or reduced, wherein the antigen is at least
one of proteins as defined above in any of (10) to (14) of [9].
[0137] [15] A tester for determining the presence or absence of a
fish antigen in an object of interest, the tester comprising an
antibody that binds to at least one of proteins as defined above in
any of (10) to (14) of [9].
[0138] [16] A tester for determining the presence or absence of an
antigen causing an allergy to fish in an object of interest, the
tester comprising a primer having a nucleotide sequence
complementary to at least one part of one of the nucleotide
sequences set forth in SEQ ID NO: 69, 109, 120, 137, or 143.
[0139] [17] A fish-derived antigen which is at least one of
proteins as defined above in any of (1) to (9) of [1] and causes an
allergy to fish.
[0140] [18] A fish-derived antigen which is at least one of
proteins as defined above in any of (10) to (14) of [9] and causes
an allergy to fish.
Advantageous Effects of Invention
[0141] The present invention can provide novel antigens of an
allergy to fish. Since the novel antigens (allergen components)
that trigger a fish allergy were identified in the present
invention, it is possible to provide highly sensitive methods and
kits for diagnosing an allergy to fish, pharmaceutical compositions
comprising such an antigen, fish or fish eggs in which the antigen
is eliminated or reduced, a processed product of the fish or the
fish eggs or fish which lay the fish eggs or have hatched from the
eggs, and testers for determining the presence or absence of a fish
antigen in an object of interest.
BRIEF DESCRIPTION OF DRAWINGS
[0142] FIG. 1A is a photograph of a gel showing a protein
electrophoretic pattern in two-dimensional electrophoresis of
proteins contained in an extract of flesh of salmon (left panel)
and a photograph of an immunoblot obtained from the two-dimensional
electrophoretic pattern by using a serum of Fish-allergic patient 1
(right panel). The bands at the left of the photograph of the gel
are bands of molecular weight markers and the numbers on the left
side of the photograph of the gel are molecular weights of
respective molecular weight markers (KDa). The numbers on the upper
side of the photograph indicate isoelectric points.
[0143] FIG. 1B is a photograph of an immunoblot obtained from the
two-dimensional electrophoretic pattern of the proteins contained
in the extract of the flesh of salmon by using a serum of
Fish-allergic patient 2 (left panel) and a photograph of an
immunoblot of the proteins stained with serum of Fish-allergic
patient 3 (right panel). The numbers on the upper side of each
photograph indicate isoelectric points.
[0144] FIG. 2 is photographs of immunoblots obtained from
two-dimensional electrophoretic patterns of proteins contained in
extracts of the flesh of 10 species of fish stained with serum of
Fish-allergic patient 1.
[0145] FIG. 3 is photographs of immunoblots obtained from
two-dimensional electrophoretic patterns of proteins contained in
extracts of the flesh of 10 species of fish stained with serum of a
subject who does not exhibit fish allergic symptoms
(Non-fish-allergic subject).
[0146] FIG. 4 is photographs of immunoblots obtained from
two-dimensional electrophoretic patterns of proteins contained in
extracts of the fish of 6 species of fish by using a serum of
Fish-allergic patient 4.
DESCRIPTION OF EMBODIMENTS
[0147] The present invention will be described in detail below, but
the present invention is not limited to them.
[0148] Unless otherwise defined herein, all scientific and
technical terms used in relation to the present invention shall
have meanings commonly understood by those skilled in the art.
[0149] As referred to herein, the "allergy" refers to the state in
which, when a certain antigen reenters a living organism sensitized
to said antigen, the living organism shows a hypersensitive
reaction detrimental to the living organism. In blood and tissues
of individuals with many food-allergic diseases, IgE antibodies
specific to antigens are produced. IgE antibodies bind to mast
cells or basophils. When an antigen specific to such an IgE
antibody reenters the body of a patient with an allergic disease,
said antigen combines with the IgE antibody bound to mast cells or
basophils, and the IgE antibody crosslinks said antigen on the cell
surface, resulting in physiological effects of IgE antibody-antigen
interaction. Examples of such physiological effects include release
of histamine, serotonin, heparin, eosinophil chemotactic factors,
leucotrienes, or the like. These released substances provoke an
allergic reaction resulting from the combination of an IgE antibody
with particular antigens. Such allergic reactions caused by
particular antigens occur through the aforementioned pathway.
[0150] As used herein, the term fish means teleost and elasmobranch
among fishes, preferably teleost, more preferably those belonging
to Salmoniformes, Perciformes, Anguilliformes, Gadiformes, and
Pleuronectiformes, further preferably those belonging to
Salmonidae, Carangidae, Congridae, Sparidae, Scombridae, Gadidae,
Anguillidae, and Paralichthyidae, and even preferably salmon, horse
mackerel, sea eel, black porgy, mackerel, sea bream, cod, young
yellowtail, eel, and flatfish. The fish may be edible.
[0151] As used herein, the term fish eggs means eggs of fish. The
fish eggs may be edible.
[0152] As referred to herein, the "allergy to fish" refers to the
state in which an individual has an allergic reaction caused by
proteins, etc. present in fish which act as an antigen. The allergy
to fish can produce an allergic reaction upon contact with, or
consumption of, an antigen present in fish. In general, allergic
reactions caused by consumption of foods are particularly referred
to as "food allergies". The allergy to fish may be a food
allergy.
[0153] As referred to herein, the "antigen" refers to a substance
that provokes an allergic reaction, and is also referred to as an
"allergen component". The antigen is preferably a protein.
[0154] As referred to herein, the "protein" refers to a molecule
having a structure in which naturally occurring amino acids are
joined together by peptide bond. The number of amino acids present
in a protein is not particularly limited, but proteins having about
2 to 50 amino acids joined together by peptide bond are in some
cases called "peptides". In the case where amino acids can form
different enantiomers, the amino acids are understood to form an
L-enantiomer, unless otherwise indicated. The amino acid sequences
of proteins or peptides as used herein are represented by
one-letter symbols of amino acids in accordance with standard usage
and the notational convention commonly used in the art. The
leftward direction represents the amino-terminal direction, and the
rightward direction represents the carboxy-terminal direction.
[0155] Identification of Antigens
[0156] Proteins contained in fish were analyzed by the
aforementioned technique to identify antigens of an allergy to
fish. To be specific, proteins were extracted from the flesh of
fish and subjected to two-dimensional electrophoresis under the
conditions described below.
[0157] The electrophoresis in the first dimension was isoelectric
focusing, which was performed using isoelectric focusing gels with
a gel-strip length of 5 to 10 cm and a gel pH range of 3 to 10. The
pH gradient of the gels in the direction of electrophoresis was as
follows: when the total gel-strip length is taken as 1, the
gel-strip length up to pH 5 is taken as "a", the gel-strip length
from pH 5 to 7 is taken as "b", and the gel-strip length above pH 7
is taken as "c", "a" is in the range of 0.15 to 0.3, "b" is in the
range of 0.4 to 0.7, and "c" is in the range of 0.15 to 0.3. More
specifically, the isoelectric focusing was performed using the IPG
gels, Immobiline Drystrip (pH 3-10 NL), produced by GE Healthcare
Bio-Sciences Corporation (hereinafter abbreviated as "GE"). The
electrophoresis system used was IPGphor produced by GE. The maximum
current of the electrophoresis system was limited to 75 .mu.A per
gel strip. The voltage program adopted to perform the
first-dimensional isoelectric focusing was as follows: (1) a
constant voltage step was performed at a constant voltage of 300 V
until the volt-hours reached 750 Vhr (the current variation width
during electrophoresis for 30 minutes before the end of this step
was 5 .mu.A); (2) the voltage was increased gradually to 1000 V for
300 Vhr; (3) the voltage was further increased gradually to 5000 V
for 4500 Vhr; and then (4) the voltage was held at a constant
voltage of 5000 V until the total Vhr reached 12000.
[0158] The electrophoresis in the second dimension was SDS-PAGE,
which was performed using polyacrylamide gels whose gel
concentration at the distal end in the direction of electrophoresis
was set to 3 to 6% and whose gel concentration at the proximal end
was set to a higher value than that at the distal end. More
specifically, the SDS-PAGE was performed using NuPAGE 4-12%
Bris-Tris Gels (IPG well, Mini, 1 mm) produced by Life
Technologies. The electrophoresis system used was XCell SureLock
Mini-Cell produced by Life Technologies. The electrophoresis was
run at a constant voltage of 200 V for about 45 minutes using an
electrophoresis buffer composed of 50 mM MOPS, 50 mM Tris base,
0.1% (w/v) SDS and 1 mM EDTA.
[0159] As a result, the following spots in a two-dimensional
electrophoresis gel run under the conditions described above for
fish proteins have been revealed to exhibit specific binding to IgE
antibodies from patients having an allergy to fish and being
diagnosed as having immediate-type allergy. [0160] Spot 1:
Molecular weight 80 to 160 kDa, pI 3.0 to 7.0 [0161] Spot 2:
Molecular weight 110 to 260 kDa, pI 4.0 to 8.0 [0162] Spot 3:
Molecular weight 80 to 160 kDa, pI 4.0 to 10.0 [0163] Spot 4:
Molecular weight 80 to 160 kDa, pI 5.0 to 11.0 [0164] Spot 5:
Molecular weight 50 to 110 kDa, pI 3.0 to 7.0 [0165] Spot 6:
Molecular weight 60 to 110 kDa, pI 4.0 to 8.0 [0166] Spot 7:
Molecular weight 40 to 80 kDa, pI 4.0 to 8.0 [0167] Spot 8:
Molecular weight 40 to 80 kDa, pI 3.0 to 7.0 [0168] Spot 9:
Molecular weight 20 to 50 kDa, pI 3.0 to 7.0 [0169] Spot 10:
Molecular weight 160 to 300 kDa, pI 3.0 to 7.0 [0170] Spot 11:
Molecular weight 80 to 160 kDa, pI 4.0 to 8.0 [0171] Spot 12:
Molecular weight 100 to 160 kDa, pI 4.0 to 7.0 [0172] Spot 13:
Molecular weight 30 to 70 kDa, pI 3.0 to 7.0 [0173] Spot 14:
Molecular weight 20 to 50 kDa, pI 5.0 to 9.0
[0174] Antigen
[0175] (1) Antigen in Spot 1
[0176] As the result of sequence identification of the antigen in
spot 1 by mass spectroscopy, the following amino acid sequences of
SEQ ID NO: 3 were detected.
[0177] Also, the mass spectroscopic data (SEQ ID NO: 3) obtained
for spot 1 on a mass spectrometer was analyzed by comparing the
data against the NCB1 protein data, and as a result, the antigen in
question was identified as Alpha-actinin-3 (amino acid sequence:
SEQ ID NO: 2, encoding nucleotide sequence: SEQ ID NO: 1). SEQ ID
NO: 3 corresponds to amino acid 572 to 585 in SEQ ID NO: 2.
[0178] Accordingly, in the present invention, the antigen in spot 1
can be any of (1A-a) to (1A-e) and (1B) as defined below: [0179]
(1A-a) a protein comprising an amino acid sequence with deletion,
substitution, insertion or addition of one or several amino acids
in SEQ ID NO: 2; [0180] (1A-b) a protein comprising an amino acid
sequence having at least 70%, preferably at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 98%, or at least 99% identity to the amino acid sequence of
SEQ ID NO: 2; [0181] (1A-c) a protein comprising an amino acid
sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 1; [0182] (1A-d) a protein comprising an amino acid
sequence encoded by a nucleotide sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the nucleotide sequence of SEQ ID NO: 1; [0183] (1A-e) a protein
comprising an amino acid sequence encoded by a nucleic acid that
hybridizes under stringent conditions with a nucleic acid having a
nucleotide sequence complementary to the nucleotide sequence of SEQ
ID NO: 1; [0184] (1B) a protein comprising at least one amino acid
sequence of SEQ ID NO: 2 or 3, preferably a protein comprising all
sequences of the amino acid sequences. As referred to above, the
amino acid sequence of SEQ ID NO: 2 or 3 may be an amino acid
sequence derived therefrom by deletion, substitution, insertion or
addition of one or several amino acids.
[0185] The proteins of (1A-a) to (1A-e) and (1B) as defined above
also include those proteins whose amino acid residues are modified
by phosphorylation, sugar chain modification, aminoacylation,
ring-opening, deamination or the like.
[0186] The proteins of (1A-a) to (1A-e) and (1B) as defined above
can be proteins that are found in a protein spot with a molecular
weight of around 80 to 160 kDa, preferably around 90 to 120 kDa and
an isoelectric point of 3.0 to 7.0, preferably an isoelectric point
of 3.0 to 6.0 on gels used in the two-dimensional electrophoresis
performed under the conditions described above in the subsection
titled "Identification of antigens".
[0187] Preferably, a protein that is an antigen in spot 1 is an
antigen of an allergy to fish.
[0188] (2) Antigen in Spot 2
[0189] As the result of sequence identification of the antigen in
spot 2 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 6-8 were detected.
[0190] Also, the mass spectroscopic data obtained for spot 2 (SEQ
ID NOs: 6-8) on a mass spectrometer was analyzed by comparing the
data against the UniProt protein data, and as a result, the antigen
in question was identified as EEF1A2 binding protein-like (amino
acid sequence: SEQ ID NO: 5, encoding nucleotide sequence: SEQ ID
NO: 4). SEQ ID NO: 6 corresponds to amino acid 143-159 in SEQ ID
NO: 5, SEQ ID NO: 7 corresponds to amino acid 395-404 in SEQ ID NO:
5, and SEQ ID NO: 8 corresponds to amino acid 427-447 in SEQ ID NO:
5.
[0191] Accordingly, in the present invention, the antigen in spot 2
can be any of (2A-a) to (2A-e) and (2B) as defined below: [0192]
(2A-a) a protein comprising an amino acid sequence with deletion,
substitution, insertion or addition of one or several amino acids
in SEQ ID NO: 5; [0193] (2A-b) a protein comprising an amino acid
sequence having at least 70%, preferably at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 98%, or at least 99% identity to the amino acid sequence of
SEQ ID NO: 5; [0194] (2A-c) a protein comprising an amino acid
sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 4; [0195] (2A-d) a protein comprising an amino acid
sequence encoded by a nucleotide sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the nucleotide sequence of SEQ ID NO: 4; [0196] (2A-e) a protein
comprising an amino acid sequence encoded by a nucleic acid that
hybridizes under stringent conditions with a nucleic acid having a
nucleotide sequence complementary to the nucleotide sequence of SEQ
ID NO: 4; [0197] (2B) a protein comprising at least one amino acid
sequence selected from the group consisting of SEQ ID NOs: 5-8,
preferably a protein comprising at least 2, 3, or all sequences of
the amino acid sequences. As referred to above, the amino acid
sequence of any of SEQ ID NOs: 5-8 may be an amino acid sequence
derived therefrom by deletion, substitution, insertion or addition
of one or several amino acids.
[0198] The proteins of (2A-a) to (2A-e) and (2B) as defined above
also include those proteins whose amino acid residues are modified
by phosphorylation, sugar chain modification, aminoacylation,
ring-opening, deamination or the like.
[0199] The proteins of (2A-a) to (2A-e) and (2B) as defined above
can be proteins that are found in a protein spot with a molecular
weight of around 110 to 260 kDa, preferably around 120 to 160 kDa
and an isoelectric point of 4.0 to 8.0, preferably an isoelectric
point of 4.0 to 7.0 on gels used in the two-dimensional
electrophoresis performed under the conditions described above in
the subsection titled "Identification of antigens".
[0200] Preferably, a protein that is an antigen in spot 2 is an
antigen of an allergy to fish.
[0201] (3) Antigen in Spot 3
[0202] As the result of sequence identification of the antigen in
spot 3 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 11-16 were detected.
[0203] Also, the mass spectroscopic data (SEQ ID NOs: 11-17)
obtained for spot 3 on a mass spectrometer was analyzed by
comparing the data against the Uniprot protein data, and as a
result, the antigen in question was identified as Alpha-1, 4-glucan
phosphorylase (amino acid sequence: SEQ ID NO: 10, encoding
nucleotide sequence: SEQ ID NO: 9). SEQ ID NO: 11 corresponds to
amino acid 836-844 in SEQ ID NO: 10, SEQ ID NO: 12 corresponds to
amino acid 13-29 in SEQ ID NO: 10, SEQ ID NO: 13 corresponds to
amino acid 657-681 in SEQ ID NO: 10, SEQ ID NO: 14 corresponds to
amino acid 471-479 in SEQ ID NO: 10, SEQ ID NO: 15 corresponds to
amino acid 546-555 in SEQ ID NO: 10, and SEQ ID NO: 16 corresponds
to amino acid 727-741 in SEQ ID NO: 10.
[0204] Accordingly, in the present invention, the antigen in spot 3
can be any of (3A-a) to (3A-e) and (3B) as defined below: [0205]
(3A-a) a protein comprising an amino acid sequence with deletion,
substitution, insertion or addition of one or several amino acids
in SEQ ID NO: 10; [0206] (3A-b) a protein comprising an amino acid
sequence having at least 70%, preferably at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 98%, or at least 99% identity to the amino acid sequence of
SEQ ID NO: 10; [0207] (3A-c) a protein comprising an amino acid
sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 9; [0208] (3A-d) a protein comprising an amino acid
sequence encoded by a nucleotide sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the nucleotide sequence of SEQ ID NO: 9. [0209] (3A-e) a protein
comprising an amino acid sequence encoded by a nucleic acid that
hybridizes under stringent conditions with a nucleic acid having a
nucleotide sequence complementary to the nucleotide sequence of SEQ
ID NO: 9; [0210] (3B) a protein comprising at least one amino acid
sequence selected from the group consisting of SEQ ID NOs: 10-16,
preferably a protein comprising at least 2, 3, 4, 5, 6 or all
sequences of the amino acid sequences. More preferably, a protein
comprising at least one amino acid sequence selected from the group
consisting of SEQ ID NOs: 10, 12, 13, 15, and 16, preferably a
protein comprising at least 2, 3, 4, or all sequences of the amino
acid sequences. As referred to above, the amino acid sequence of
any of SEQ ID NOs: 10-16 may be an amino acid sequence derived
therefrom by deletion, substitution, insertion or addition of one
or several amino acids.
[0211] The proteins of (3A-a) to (3A-e) and (3B) as defined above
also include those proteins whose amino acid residues are modified
by phosphorylation, sugar chain modification, aminoacylation,
ring-opening, deamination or the like.
[0212] The proteins of (3A-a) to (3A-e) and (3B) as defined above
can be proteins that are found in a protein spot with a molecular
weight of around 80 to 160 kDa, preferably around 90 to 120 kDa and
an isoelectric point of 4.0 to 10.0, preferably an isoelectric
point of 5.0 to 8.0 on gels used in the two-dimensional
electrophoresis performed under the conditions described above in
the subsection titled "Identification of antigens".
[0213] Preferably, a protein that is an antigen in spot 3 is an
antigen of an allergy to fish.
[0214] (4) Antigen in Spot 4
[0215] As the result of sequence identification of the antigen in
spot 4 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 20-24 were detected.
[0216] Also, the mass spectroscopic data (SEQ ID NOs: 20-24)
obtained for spot 4 on a mass spectrometer was analyzed by
comparing the data against the Uniprot protein data, and as a
result, the antigen in question was identified as Elongation factor
2 (amino acid sequence: SEQ ID NO: 19, encoding nucleotide
sequence: SEQ ID NO: 18). SEQ ID NO: 20 corresponds to amino acid
831-840 in SEQ ID NO: 19, SEQ ID NO: 21 corresponds to amino acid
560-571 in SEQ ID NO: 19, SEQ ID NO: 22 corresponds to amino acid
560-572 in SEQ ID NO: 19, SEQ ID NO: 23 corresponds to amino acid
677-688 in SEQ ID NO: 19, and SEQ ID NO: 24 corresponds to amino
acid 620-629 in SEQ ID NO: 19.
[0217] Accordingly, in the present invention, the antigen in spot 4
can be any of (4-a) to (4-e) and (4B) as defined below: [0218]
(4A-a) a protein comprising an amino acid sequence with deletion,
substitution, insertion or addition of one or several amino acids
in SEQ ID NO: 19; [0219] (4A-b) a protein comprising an amino acid
sequence having at least 70%, preferably at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 98%, or at least 99% identity to the amino acid sequence of
SEQ ID NO: 19; [0220] (4A-c) a protein comprising an amino acid
sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 18; [0221] (4A-d) a protein comprising an amino acid
sequence encoded by a nucleotide sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the nucleotide sequence of SEQ ID NO: 18; [0222] (4A-e) a
protein comprising an amino acid sequence encoded by a nucleic acid
that hybridizes under stringent conditions with a nucleic acid
having a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 18; [0223] (4B) a protein comprising at
least one amino acid sequence selected from the group consisting of
SEQ ID NOs: 19-24, preferably a protein comprising at least 2, 3,
4, 5, 6 or all sequences of the amino acid sequences. As referred
to above, the amino acid sequence of any of SEQ ID NOs: 19-24 may
be an amino acid sequence derived therefrom by deletion,
substitution, insertion or addition of one or several amino
acids.
[0224] The proteins of (4A-a) to (4A-e) and (4B) as defined above
also include those proteins whose amino acid residues are modified
by phosphorylation, sugar chain modification, aminoacylation,
ring-opening, deamination or the like.
[0225] The proteins of (4A-a) to (4A-e) and (4B) as defined above
can be proteins that are found in a protein spot with a molecular
weight of around 80 to 160 kDa, preferably around 90 to 120 kDa and
an isoelectric point of 5.0 to 11.0, preferably an isoelectric
point of 6.0 to 8.0 on gels used in the two-dimensional
electrophoresis performed under the conditions described above in
the subsection titled "Identification of antigens".
[0226] Preferably, a protein that is an antigen in spot 4 is an
antigen of an allergy to fish.
[0227] (5) Antigen in Spot 5
[0228] As the result of sequence identification of the antigen in
spot 5 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 27-31 were detected.
[0229] Also, the mass spectroscopic data (SEQ ID NOs: 27-31)
obtained for spot 5 on a mass spectrometer was analyzed by
comparing the data against the Uniprot protein data, and as a
result, the antigen in question was identified as Heat shock
cognate 70 kDa protein (amino acid sequence: SEQ ID NO: 26,
encoding nucleotide sequence: SEQ ID NO: 25). SEQ ID NO: 27
corresponds to amino acid 113-126 in SEQ ID NO: 26, SEQ ID NO: 28
corresponds to amino acid 513-526 in SEQ ID NO: 26, SEQ ID NO: 29
corresponds to amino acid 89-102 in SEQ ID NO: 26, SEQ ID NO: 30
corresponds to amino acid 584-597 in SEQ ID NO: 26, and SEQ ID NO:
31 corresponds to amino acid 138-159 in SEQ ID NO: 26.
[0230] Accordingly, in the present invention, the antigen in spot 5
can be any of (5A-a) to (5A-e) and (5B) as defined below: [0231]
(5A-a) a protein comprising an amino acid sequence with deletion,
substitution, insertion or addition of one or several amino acids
in SEQ ID NO: 26; [0232] (5A-b) a protein comprising an amino acid
sequence having at least 70%, preferably at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 98%, or at least 99% identity to the amino acid sequence of
SEQ ID NO: 26; [0233] (5A-c) a protein comprising an amino acid
sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 25; [0234] (5A-d) a protein comprising an amino acid
sequence encoded by a nucleotide sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the nucleotide sequence of SEQ ID NO: 25; [0235] (5A-e) a
protein comprising an amino acid sequence encoded by a nucleic acid
that hybridizes under stringent conditions with a nucleic acid
having a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 25; [0236] (5B) a protein comprising at
least one amino acid sequence selected from the group consisting of
SEQ ID NOs: 26-31, preferably a protein comprising at least 2, 3,
4, 5 or all sequences of the amino acid sequences. As referred to
above, the amino acid sequence of any of SEQ ID NOs: 26-31 may be
an amino acid sequence derived therefrom by deletion, substitution,
insertion or addition of one or several amino acids.
[0237] The proteins of (5A-a) to (5A-e) and (5B) as defined above
also include those proteins whose amino acid residues are modified
by phosphorylation, sugar chain modification, aminoacylation,
ring-opening, deamination or the like.
[0238] The proteins of (5A-a) to (5A-e) and (5B) as defined above
can be proteins that are found in a protein spot with a molecular
weight of around 50 to 110 kDa, preferably around 60 to 90 kDa and
an isoelectric point of 3.0 to 7.0, preferably an isoelectric point
of 3.0 to 6.0 on gels used in the two-dimensional electrophoresis
performed under the conditions described above in the subsection
titled "Identification of antigens".
[0239] Preferably, a protein that is an antigen is an antigen of an
allergy to fish.
[0240] (6) Antigen in Spot 6
[0241] As the result of sequence identification of the antigen in
spot 6 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 34-41 were detected.
[0242] Also, the mass spectroscopic data (SEQ ID NOs: 34-41)
obtained for spot 6 on a mass spectrometer was analyzed by
comparing the data against the Uniprot protein data, and as a
result, the antigen in question was identified as Serotransferrin
(amino acid sequence: SEQ ID NO: 33, encoding nucleotide sequence:
SEQ ID NO: 32). SEQ ID NO: 34 corresponds to amino acid 330-341 in
SEQ ID NO: 33, SEQ ID NO: 35 corresponds to amino acid 332-341 in
SEQ ID NO: 33, SEQ ID NO: 36 corresponds to amino acid 264-274 in
SEQ ID NO: 33, SEQ ID NO: 37 corresponds to amino acid 113-124 in
SEQ ID NO: 33, SEQ ID NO: 38 corresponds to amino acid 112-124 in
SEQ ID NO: 33, SEQ ID NO: 39 corresponds to amino acid 112-125 in
SEQ ID NO: 33, SEQ ID NO: 40 corresponds to amino acid 275-292 in
SEQ ID NO: 33, and SEQ ID NO: 41 corresponds to amino acid 436-446
in SEQ ID NO: 33.
[0243] Accordingly, in the present invention, the antigen in spot 6
can be any of (6A-a) to (6A-e) and (6B) as defined below: [0244]
(6A-a) a protein comprising an amino acid sequence with deletion,
substitution, insertion or addition of one or several amino acids
in SEQ ID NO: 33; [0245] (6A-b) a protein comprising an amino acid
sequence having at least 70%, preferably at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 98%, or at least 99% identity to the amino acid sequence of
SEQ ID NO: 33; [0246] (6A-c) a protein comprising an amino acid
sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 32; [0247] (6A-d) a protein comprising an amino acid
sequence encoded by a nucleotide sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the nucleotide sequence of SEQ ID NO: 32; [0248] (6A-e) a
protein comprising an amino acid sequence encoded by a nucleic acid
that hybridizes under stringent conditions with a nucleic acid
having a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 32; [0249] (6B) a protein comprising at
least one amino acid sequence selected from the group consisting of
SEQ ID NOs: 33-41, preferably a protein comprising at least 2, 3,
4, 5, 6, 7, 8 or all sequences of the amino acid sequences. As
referred to above, the amino acid sequence of any of SEQ ID NOs:
33-41 may be an amino acid sequence derived therefrom by deletion,
substitution, insertion or addition of one or several amino
acids.
[0250] The proteins of (6A-a) to (6A-e) and (6B) as defined above
also include those proteins whose amino acid residues are modified
by phosphorylation, sugar chain modification, aminoacylation,
ring-opening, deamination or the like.
[0251] The proteins of (6A-a) to (6A-e) and (6B) as defined above
can be proteins that are found in a protein spot with a molecular
weight of around 60 to 110 kDa, preferably around 70 to 100 kDa and
an isoelectric point of 4.0 to 8.0, preferably an isoelectric point
of 5.0 to 7.0 on gels used in the two-dimensional electrophoresis
performed under the conditions described above in the subsection
titled "Identification of antigens".
[0252] Preferably, a protein that is an antigen in spot 6 is an
antigen of an allergy to fish.
[0253] (7) Antigen in Spot 7
[0254] As the result of sequence identification of the antigen in
spot 7 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 44-54 were detected.
[0255] Also, the mass spectroscopic data (SEQ ID NOs: 44-54)
obtained for spot 7 on a mass spectrometer was analyzed by
comparing the data against the Uniprot protein data, and as a
result, the antigen in question was identified as Myosin binding
protein H-like (amino acid sequence: SEQ ID NO: 43, encoding
nucleotide sequence: SEQ ID NO: 42). SEQ ID NO: 44 corresponds to
amino acid 275-292 in SEQ ID NO: 43, SEQ ID NO: 45 corresponds to
amino acid 49-66 in SEQ ID NO: 43, SEQ ID NO: 46 corresponds to
amino acid 67-77 in SEQ ID NO: 43, SEQ ID NO: 47 corresponds to
amino acid 310-323 in SEQ ID NO: 43, SEQ ID NO: 48 corresponds to
amino acid 382-401 in SEQ ID NO: 43, SEQ ID NO: 49 corresponds to
amino acid 402-418 in SEQ ID NO: 43, SEQ ID NO: 50 corresponds to
amino acid 210-219 in SEQ ID NO: 43, SEQ ID NO: 51 corresponds to
amino acid 210-228 in SEQ ID NO: 43, SEQ ID NO: 52 corresponds to
amino acid 152-183 in SEQ ID NO: 43, SEQ ID NO: 53 corresponds to
amino acid 100-112 in SEQ ID NO: 43, and SEQ ID NO: 54 corresponds
to amino acid 113-129 in SEQ ID NO: 43.
[0256] Accordingly, in the present invention, the antigen in spot 7
can be any of (7A-a) to (7A-e) and (7B) as defined below: [0257]
(7A-a) a protein comprising an amino acid sequence with deletion,
substitution, insertion or addition of one or several amino acids
in SEQ ID NO: 43; [0258] (7A-b) a protein comprising an amino acid
sequence having at least 70%, preferably at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 98%, or at least 99% identity to the amino acid sequence of
SEQ ID NO: 43; [0259] (7A-c) a protein comprising an amino acid
sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 42; [0260] (7A-d) a protein comprising an amino acid
sequence encoded by a nucleotide sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the nucleotide sequence of SEQ ID NO: 42; [0261] (7A-e) a
protein comprising an amino acid sequence encoded by a nucleic acid
that hybridizes under stringent conditions with a nucleic acid
having a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 42; [0262] (7B) a protein comprising at
least one amino acid sequence selected from the group consisting of
SEQ ID NOs: 43-54, preferably a protein comprising at least 2, 3,
4, 5, 6, 7, 8, 9, 10, 11 or all sequences of the amino acid
sequences; As referred to above, the amino acid sequence of any of
SEQ ID NOs: 43-54 may be an amino acid sequence derived therefrom
by deletion, substitution, insertion or addition of one or several
amino acids.
[0263] The proteins of (7A-a) to (7A-e) and (7B) as defined above
also include those proteins whose amino acid residues are modified
by phosphorylation, sugar chain modification, aminoacylation,
ring-opening, deamination or the like.
[0264] The proteins of (7A-a) to (7A-e) and (7B) as defined above
can be proteins that are found in a protein spot with a molecular
weight of around 40 to 80 kDa, preferably around 50 to 60 kDa and
an isoelectric point of 4.0 to 8.0, preferably an isoelectric point
of 4.0 to 7.0 on gels used in the two-dimensional electrophoresis
performed under the conditions described above in the subsection
titled "Identification of antigens".
[0265] Preferably, a protein that is an antigen in spot 7 is an
antigen of an allergy to fish.
[0266] (8) Antigen in Spot 8
[0267] As the result of sequence identification of the antigen in
spot 8 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 57-59 were detected.
[0268] Also, the mass spectroscopic data (SEQ ID NOs: 57-59)
obtained for spot 8 on a mass spectrometer was analyzed by
comparing the data against the Uniprot protein data, and as a
result, the antigen in question was identified as Desmin (Fragment)
(amino acid sequence: SEQ ID NO: 56, encoding nucleotide sequence:
SEQ ID NO: 55). SEQ ID NO: 57 corresponds to amino acid 364-380 in
SEQ ID NO: 56, SEQ ID NO: 58 corresponds to amino acid 74-94 in SEQ
ID NO: 56, and SEQ ID NO: 59 corresponds to amino acid 273-282 in
SEQ ID NO: 56.
[0269] Accordingly, in the present invention, the antigen in spot 8
can be any of (8A-a) to (8A-e) and (8B) as defined below: [0270]
(8A-a) a protein comprising an amino acid sequence with deletion,
substitution, insertion or addition of one or several amino acids
in SEQ ID NO: 56; [0271] (8A-b) a protein comprising an amino acid
sequence having at least 70%, preferably at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 98%, or at least 99% identity to the amino acid sequence of
SEQ ID NO: 56; [0272] (8A-c) a protein comprising an amino acid
sequence encoded by a nucleotide sequence with deletion,
substitution, insertion or addition of one or several nucleotides
in SEQ ID NO: 55; [0273] (8A-d) a protein comprising an amino acid
sequence encoded by a nucleotide sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the nucleotide sequence of SEQ ID NO: 55; [0274] (8A-e) a
protein comprising an amino acid sequence encoded by a nucleic acid
that hybridizes under stringent conditions with a nucleic acid
having a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 55; [0275] (8B) a protein comprising at
least one amino acid sequence selected from the group consisting of
SEQ ID NOs: 56-59, preferably a protein comprising at least 2, 3,
or all sequences of the amino acid sequences. As referred to above,
the amino acid sequence of any of SEQ ID NOs: 56-59 may be an amino
acid sequence derived therefrom by deletion, substitution,
insertion or addition of one or several amino acids.
[0276] The proteins of (8A-a) to (8A-e) and (8B) as defined above
also include those proteins whose amino acid residues are modified
by phosphorylation, sugar chain modification, aminoacylation,
ring-opening, deamination or the like.
[0277] The proteins of (8A-a) to (8A-e) and (8B) as defined above
can be proteins that are found in a protein spot with a molecular
weight of around 40 to 80 kDa, preferably around 50 to 60 kDa and
an isoelectric point of 3.0 to 7.0, preferably an isoelectric point
of 4.0 to 6.0 on gels used in the two-dimensional electrophoresis
performed under the conditions described above in the subsection
titled "Identification of antigens".
[0278] Preferably, a protein that is an antigen in spot 8 is an
antigen of an allergy to fish.
[0279] (9) Antigen in Spot 9
[0280] As the result of sequence identification of the antigen in
spot 9 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 62-68 were detected.
[0281] Also, the mass spectroscopic data (SEQ ID NOs: 62-68)
obtained for spot 9 on a mass spectrometer was analyzed by
comparing the data against the UniProt protein data, and as a
result, the antigen in question was identified as Capping protein
(Actin filament) muscle Z-line beta (amino acid sequence: SEQ ID
NO: 61, encoding nucleotide sequence: SEQ ID NO: 60). SEQ ID NO: 62
corresponds to amino acid 200-223 in SEQ ID NO: 61, SEQ ID NO: 63
corresponds to amino acid 238-254 in SEQ ID NO: 61, SEQ ID NO: 64
corresponds to amino acid 261-274 in SEQ ID NO: 61, SEQ ID NO: 65
corresponds to amino acid 224-235 in SEQ ID NO: 61, SEQ ID NO: 66
corresponds to amino acid 169-181 in SEQ ID NO: 61, SEQ ID NO: 67
corresponds to amino acid 245-254 in SEQ ID NO: 61, and SEQ ID NO:
68 corresponds to amino acid 79-95 in SEQ ID NO: 61.
[0282] Accordingly, the antigen in spot 9 in the present invention
can be any of the proteins as defined below in (9A-a) to (9A-e),
and (9B): [0283] (9A-a) a protein comprising an amino acid sequence
with deletion, substitution, insertion or addition of one or
several amino acids in SEQ ID NO: 61; [0284] (9A-b) a protein
comprising an amino acid sequence having at least 70%, preferably
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at least 98%, or at least 99% identity to the
amino acid sequence of SEQ ID NO: 61; [0285] (9A-c) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
with deletion, substitution, insertion or addition of one or
several nucleotides in SEQ ID NO: 60; [0286] (9A-d) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
having at least 70%, preferably at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%,
or at least 99% identity to the nucleotide sequence of SEQ ID NO:
60; [0287] (9A-e) a protein comprising an amino acid sequence
encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 60; [0288]
(9B) a protein comprising at least one amino acid sequence selected
from the group consisting of SEQ ID NOs: 61-68, preferably a
protein comprising at least 2, 3, 4, 5, 6, 7 or all sequences of
the amino acid sequences. As referred to above, the amino acid
sequence of any of SEQ ID NOs: 61-68 may be an amino acid sequence
derived therefrom by deletion, substitution, insertion or addition
of one or several amino acids;
[0289] The proteins as defined above in (9A-a) to (9A-e), and (9B)
also include those proteins whose amino acid residues are modified
by phosphorylation, sugar chain modification, aminoacylation,
ring-opening, deamination or the like.
[0290] The proteins as defined above in (9A-a) to (9A-e), and (9B)
can be proteins that are found in a protein spot with a molecular
weight of around 20 to 50 kDa, preferably around 30 to 40 kDa, and
an isoelectric point of 3.0 to 7.0, preferably an isoelectric point
of 4.0 to 6.0 on gels used in the two-dimensional electrophoresis
performed under the conditions described above in the subsection
titled "Identification of antigens".
[0291] Preferably, a protein that is an antigen in spot 9 is an
antigen of an allergy to fish.
[0292] (10) Antigen in Spot 10
[0293] As the result of sequence identification of the antigen in
spot 10 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 71-108 were detected.
[0294] Also, the mass spectroscopic data (SEQ ID NOs: 71-108)
obtained for spot 10 on a mass spectrometer was analyzed by
comparing the data against the NCBI protein data, and as a result,
the antigen in question was identified as myosin heavy chain, fast
skeletal muscle-like (amino acid sequence: SEQ ID NO: 70, encoding
nucleotide sequence: SEQ ID NO: 69). SEQ ID NO: 71 corresponds to
amino acid 13-19 in SEQ ID NO:70, SEQ ID NO: 72 corresponds to
amino acid 262-271 in SEQ ID NO: 70, SEQ ID NO: 73 corresponds to
amino acid 996-1014 in SEQ ID NO: 70, SEQ ID NO: 74 corresponds to
amino acid 951-961 in SEQ ID NO: 70, SEQ ID NO: 75 corresponds to
amino acid 1293-1303 in SEQ ID NO: 70, SEQ ID NO: 76 corresponds to
amino acid 1082-1091 in SEQ ID NO: 70, SEQ ID NO: 77 corresponds to
amino acid 1847-1856 in SEQ ID NO:70, SEQ ID NO: 78 corresponds to
amino acid 172-186 in SEQ ID NO: 70, SEQ ID NO: 79 corresponds to
amino acid 919-937 in SEQ ID NO: 70, SEQ ID NO: 80 corresponds to
amino acid 249-258 in SEQ ID NO: 70, SEQ ID NO: 81 corresponds to
amino acid 706-717 in SEQ ID NO: 70, SEQ ID NO: 82 corresponds to
amino acid 50-59 in SEQ ID NO: 70, SEQ ID NO: 83 corresponds to
amino acid 415-430 in SEQ ID NO:70, SEQ ID NO: 84 corresponds to
amino acid 834-845 in SEQ ID NO: 70, SEQ ID NO: 85 corresponds to
amino acid 617-630 in SEQ ID NO: 70, SEQ ID NO: 86 corresponds to
amino acid 1556-1567 in SEQ ID NO: 70, SEQ ID NO: 87 corresponds to
amino acid 1391-1408 in SEQ ID NO: 70, SEQ ID NO: 88 corresponds to
amino acid 1025-1040 in SEQ ID NO: 70, SEQ ID NO: 89 corresponds to
amino acid 1813-1836 in SEQ ID NO:70, SEQ ID NO: 90 corresponds to
amino acid 743-755 in SEQ ID NO: 70, SEQ ID NO: 91 corresponds to
amino acid 1172-1192 in SEQ ID NO: 70, SEQ ID NO: 92 corresponds to
amino acid 353-364 in SEQ ID NO: 70, SEQ ID NO: 93 corresponds to
amino acid 1261-1292 in SEQ ID NO: 70, SEQ ID NO: 94 corresponds to
amino acid 1783-1789 in SEQ ID NO: 70, SEQ ID NO: 95 corresponds to
amino acid 1502-1519 in SEQ ID NO: 70, SEQ ID NO: 96 corresponds to
amino acid 1484-1497 in SEQ ID NO:70, SEQ ID NO: 97 corresponds to
amino acid 1194-1212 in SEQ ID NO: 70, SEQ ID NO: 98 corresponds to
amino acid 1304-1314 in SEQ ID NO: 70, SEQ ID NO: 99 corresponds to
amino acid 369-384 in SEQ ID NO: 70, SEQ ID NO: 100 corresponds to
amino acid 1315-1322 in SEQ ID NO: 70, SEQ ID NO: 101 corresponds
to amino acid 1536-1555 in SEQ ID NO: 70, SEQ ID NO: 102
corresponds to amino acid 237-248 in SEQ ID NO:70, SEQ ID NO: 103
corresponds to amino acid 1699-1725 in SEQ ID NO: 70, SEQ ID NO:
104 corresponds to amino acid 1092-1104 in SEQ ID NO: 70, SEQ ID
NO: 105 corresponds to amino acid 407-414 in SEQ ID NO: 70, SEQ ID
NO: 106 corresponds to amino acid 1897-1917 in SEQ ID NO: 70, SEQ
ID NO:107 corresponds to amino acid 1458-1470 in SEQ ID NO: 70, and
SEQ ID NO: 108 corresponds to amino acid 1373-1388 in SEQ ID NO:
70.
[0295] Accordingly, the antigen in spot 10 in the present invention
can be any of the proteins as defined below in (10A-a) to (10A-e),
and (10B): [0296] (10A-a) a protein comprising an amino acid
sequence with deletion, substitution, insertion or addition of one
or several amino acids in SEQ ID NO: 70; [0297] (10A-b) a protein
comprising an amino acid sequence having at least 70%, preferably
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at least 98%, or at least 99% identity to the
amino acid sequence of SEQ ID NO: 70; [0298] (10A-c) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
with deletion, substitution, insertion or addition of one or
several nucleotides in SEQ ID NO: 69; [0299] (10A-d) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
having at least 70%, preferably at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%,
or at least 99% identity to the nucleotide sequence of SEQ ID NO:
69; [0300] (10A-e) a protein comprising an amino acid sequence
encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 69; [0301]
(10B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 70-108,
preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or all sequences of
the amino acid sequences. As referred to above, the amino acid
sequence of any of SEQ ID NOs: 70-108 may be an amino acid sequence
derived therefrom by deletion, substitution, insertion or addition
of one or several amino acids;
[0302] The proteins as defined above in (10A-a) to (10A-e), and
(10B) also include those proteins whose amino acid residues are
modified by phosphorylation, sugar chain modification,
aminoacylation, ring-opening, deamination or the like.
[0303] The proteins as defined above in (10A-a) to (10A-e), and
(10B) can be proteins that are found in a protein spot with a
molecular weight of around 160 to 300 kDa, preferably around 160 to
260 kDa, more preferably around 200 to 230 kDa, and an isoelectric
point of 3.0 to 7.0, preferably an isoelectric point of 4.0 to 6.0,
more preferably an isoelectric point of 4.5 to 5.5 on gels used in
the two-dimensional electrophoresis performed under the conditions
described above in the subsection titled "Identification of
antigens".
[0304] Preferably, a protein that is an antigen in spot 10 is an
antigen of an allergy to fish.
[0305] (11) Antigen in Spot 11
[0306] As the result of sequence identification of the antigen in
spot 11 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 111-119 were detected.
[0307] Also, the mass spectroscopic data (SEQ ID NOs: 111-119)
obtained for spot 11 on a mass spectrometer was analyzed by
comparing the data against the NCBI protein data, and as a result,
the antigen in question was identified as glycogen phosphorylase,
muscle form-like (amino acid sequence: SEQ ID NO: 110, encoding
nucleotide sequence: SEQ ID NO: 109). SEQ ID NO: 111 corresponds to
amino acid 471-479 in SEQ ID NO: 110, SEQ ID NO: 112 corresponds to
amino acid 547-555 in SEQ ID NO: 110, SEQ ID NO: 113 corresponds to
amino acid 203-215 in SEQ ID NO:110, SEQ ID NO: 114 corresponds to
amino acid 727-741 in SEQ ID NO: 110, SEQ ID NO: 115 corresponds to
amino acid 508-520 in SEQ ID NO: 110, SEQ ID NO: 116 corresponds to
amino acid 742-755 in SEQ ID NO: 110, SEQ ID NO: 117 corresponds to
amino acid 13-29 in SEQ ID NO: 110, SEQ ID NO:118 corresponds to
amino acid 775-784 in SEQ ID NO: 110, and SEQ ID NO: 119
corresponds to amino acid 643-656 in SEQ ID NO: 110.
[0308] Accordingly, the antigens in spot 11 in the present
invention can be any of the proteins as defined below in (11A-a) to
(11A-e) and (11B): [0309] (11A-a) a protein comprising an amino
acid sequence with deletion, substitution, insertion or addition of
one or several amino acids in SEQ ID NO: 110; [0310] (11A-b) a
protein comprising an amino acid sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the amino acid sequence of SEQ ID NO: 110; [0311] (11A-c) a
protein comprising an amino acid sequence encoded by a nucleotide
sequence with deletion, substitution, insertion or addition of one
or several nucleotides in SEQ ID NO: 109; [0312] (11A-d) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
having at least 70%, preferably at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%,
or at least 99% identity to the nucleotide sequence of SEQ ID NO:
109; [0313] (11A-e) a protein comprising an amino acid sequence
encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 109; [0314]
(11B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 110-119,
preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9 or
all sequences of the amino acid sequences. As referred to above,
the amino acid sequence of any of SEQ ID NOs: 110-119 may be an
amino acid sequence derived therefrom by deletion, substitution,
insertion or addition of one or several amino acids.
[0315] The proteins of (11A-a) to (11A-e) and (11B) as defined
above also include those proteins whose amino acid residues are
modified by phosphorylation, sugar chain modification,
aminoacylation, ring-opening, deamination or the like.
[0316] The proteins of (11A-a) to (11A-e) and (11B) as defined
above can be proteins that are found in a protein spot with a
molecular weight of around 80 to 160 kDa, preferably around 80 to
110 kDa, more preferably around 90 to 110 kDa and an isoelectric
point of 4.0 to 8.0, preferably an isoelectric point of 5.0 to 7.5,
more preferably an isoelectric point of 6.5 to 7.0 on gels used in
the two-dimensional electrophoresis performed under the conditions
described above in the subsection titled "Identification of
antigens".
[0317] Preferably, a protein that is an antigen in spot 11 is an
antigen of an allergy to fish.
[0318] (12) Antigen in Spot 12
[0319] As the result of sequence identification of the antigen in
spot 12 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 122-136 were detected.
[0320] Also, the mass spectroscopic data (SEQ ID NOs: 122-136)
obtained for spot 12 on a mass spectrometer was analyzed by
comparing the data against the NCBI protein data, and as a result,
the antigen in question was identified as myosin-binding protein C,
fast-type-like (amino acid sequence: SEQ ID NO: 121, encoding
nucleotide sequence: SEQ ID NO: 120). SEQ ID NO: 122 corresponds to
amino acid 197-204 in SEQ ID NO: 121, SEQ ID NO: 123 corresponds to
amino acid 421-434 in SEQ ID NO: 121, SEQ ID NO: 124 corresponds to
amino acid 329-336 in SEQ ID NO:121, SEQ ID NO: 125 corresponds to
amino acid 413-420 in SEQ ID NO: 121, SEQ ID NO:126 corresponds to
amino acid 240-246 in SEQ ID NO: 121, SEQ ID NO: 127 corresponds to
amino acid 214-228 in SEQ ID NO: 121, SEQ ID NO: 128 corresponds to
amino acid 1014-1024 in SEQ ID NO: 121, SEQ ID NO: 129 corresponds
to amino acid 842-855 in SEQ ID NO: 121, SEQ ID NO: 130 corresponds
to amino acid 260-274 in SEQ ID NO:121, SEQ ID NO: 131 corresponds
to amino acid 672-685 in SEQ ID NO: 121, SEQ ID NO: 132 corresponds
to amino acid 506-512 in SEQ ID NO: 121, SEQ ID NO: 133 corresponds
to amino acid 205-213 in SEQ ID NO: 121, SEQ ID NO: 134 corresponds
to amino acid 165-182 in SEQ ID NO: 121, SEQ ID NO:135 corresponds
to amino acid 321-328 in SEQ ID NO: 121, and SEQ ID NO: 136
corresponds to amino acid 70-82 in SEQ ID NO: 121.
[0321] Accordingly, the antigens in spot 12 in the present
invention can be any of the proteins as defined below in (12A-a) to
(12A-e) and (12B): [0322] (12A-a) a protein comprising an amino
acid sequence with deletion, substitution, insertion or addition of
one or several amino acids in SEQ ID NO: 121; [0323] (12A-b) a
protein comprising an amino acid sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the amino acid sequence of SEQ ID NO: 121; [0324] (12A-c) a
protein comprising an amino acid sequence encoded by a nucleotide
sequence with deletion, substitution, insertion or addition of one
or several nucleotides in SEQ ID NO: 119; [0325] (12A-d) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
having at least 70%, preferably at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%,
or at least 99% identity to the nucleotide sequence of SEQ ID NO:
119; [0326] (12A-e) a protein comprising an amino acid sequence
encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 119; [0327]
(12B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 121-136,
preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15 or all sequences of the amino acid
sequences. As referred to above, the amino acid sequence of any of
SEQ ID NOs: 121-136 may be an amino acid sequence derived therefrom
by deletion, substitution, insertion or addition of one or several
amino acids.
[0328] The proteins of (12A-a) to (12A-e) and (12B) as defined
above also include those proteins whose amino acid residues are
modified by phosphorylation, sugar chain modification,
aminoacylation, ring-opening, deamination or the like.
[0329] The proteins of (12A-a) to (12A-e) and (12B) as defined
above can be proteins that are found in a protein spot with a
molecular weight of around 100 to 160 kDa, preferably around 110 to
150 kDa, more preferably around 120 to 140 kDa and an isoelectric
point of 4.0 to 7.0, preferably an isoelectric point of 4.0 to 6.0,
more preferably an isoelectric point of 5.0 to 6.0 on gels used in
the two-dimensional electrophoresis performed under the conditions
described above in the subsection titled "Identification of
antigens".
[0330] Preferably, a protein that is an antigen in spot 12 is an
antigen of an allergy to fish.
[0331] (13) Antigen in Spot 13
[0332] As the result of sequence identification of the antigen in
spot 13 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 139-142 were detected.
[0333] Also, the mass spectroscopic data (SEQ ID NOs: 139-142)
obtained for spot 13 on a mass spectrometer was analyzed by
comparing the data against the NCBI protein data, and as a result,
the antigen in question was identified as ATP synthase subunit
beta, mitochondrial (amino acid sequence: SEQ ID NO: 138, encoding
nucleotide sequence: SEQ ID NO: 137). SEQ ID NO: 139 corresponds to
amino acid 191-201 in SEQ ID NO: 138, SEQ ID NO: 140 corresponds to
amino acid 449-469 in SEQ ID NO: 138, SEQ ID NO:141 corresponds to
amino acid 202-214 in SEQ ID NO: 138, and SEQ ID NO: 142
corresponds to amino acid 178-187 in SEQ ID NO: 138.
[0334] Accordingly, the antigen in spot 13 in the present invention
can be any of the proteins as defined below in (13A-a) to (13A-e),
and (13B): [0335] (13A-a) a protein comprising an amino acid
sequence with deletion, substitution, insertion or addition of one
or several amino acids in SEQ ID NO: 138; [0336] (13A-b) a protein
comprising an amino acid sequence having at least 70%, preferably
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at least 98%, or at least 99% identity to the
amino acid sequence of SEQ ID NO: 138; [0337] (13A-c) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
with deletion, substitution, insertion or addition of one or
several nucleotides in SEQ ID NO: 137; [0338] (13A-d) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
having at least 70%, preferably at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%,
or at least 99% identity to the nucleotide sequence of SEQ ID NO:
137; [0339] (13A-e) a protein comprising an amino acid sequence
encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 137; [0340]
(13B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 138-142,
preferably a protein comprising at least 2, 3, 4 or all sequences
of the amino acid sequences. As referred to above, the amino acid
sequence of any of SEQ ID NOs: 138-142 may be an amino acid
sequence derived therefrom by deletion, substitution, insertion or
addition of one or several amino acids;
[0341] The proteins as defined above in (13A-a) to (13A-e), and
(13B) also include those proteins whose amino acid residues are
modified by phosphorylation, sugar chain modification,
aminoacylation, ring-opening, deamination or the like.
[0342] The proteins as defined above in (13A-a) to (13A-e), and
(13B) can be proteins that are found in a protein spot with a
molecular weight of around 30 to 70 kDa, preferably around 40 to 60
kDa, more preferably around 45 to 55 kDa and an isoelectric point
of 3.0 to 7.0, preferably an isoelectric point of 3.0 to 6.0, more
preferably an isoelectric point of 4.0 to 5.5 on gels used in the
two-dimensional electrophoresis performed under the conditions
described above in the subsection titled "Identification of
antigens".
[0343] Preferably, a protein that is an antigen in spot 13 is an
antigen of an allergy to fish.
[0344] (14) Antigen in Spot 14
[0345] As the result of sequence identification of the antigen in
spot 14 by mass spectroscopy, the following amino acid sequences of
SEQ ID NOs: 145-149 were detected.
[0346] Also, the mass spectroscopic data (SEQ ID NOs: 145-149)
obtained for spot 14 on a mass spectrometer was analyzed by
comparing the data against the NCBI protein data, and as a result,
the antigen in question was identified as L-lactate dehydrogenase A
chain-like (amino acid sequence: SEQ ID NO: 144, encoding
nucleotide sequence: SEQ ID NO: 143). SEQ ID NO: 145 corresponds to
amino acid 119-126 in SEQ ID NO: 144, SEQ ID NO: 146 corresponds to
amino acid 7-22 in SEQ ID NO: 144, SEQ ID NO: 147 corresponds to
amino acid 9-22 in SEQ ID NO: 144, SEQ ID NO: 148 corresponds to
amino acid 270-278 in SEQ ID NO: 144, and SEQ ID NO: 149
corresponds to amino acid 91-118 in SEQ ID NO: 144.
[0347] Accordingly, the antigens in spot 14 in the present
invention can be any of the proteins as defined below in (14A-a) to
(14A-e) and (14B): [0348] (14A-a) a protein comprising an amino
acid sequence with deletion, substitution, insertion or addition of
one or several amino acids in SEQ ID NO: 144; [0349] (14A-b) a
protein comprising an amino acid sequence having at least 70%,
preferably at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 98%, or at least 99% identity
to the amino acid sequence of SEQ ID NO: 144; [0350] (14A-c) a
protein comprising an amino acid sequence encoded by a nucleotide
sequence with deletion, substitution, insertion or addition of one
or several nucleotides in SEQ ID. NO: 143; [0351] (14A-d) a protein
comprising an amino acid sequence encoded by a nucleotide sequence
having at least 70%, preferably at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 98%,
or at least 99% identity to the nucleotide sequence of SEQ ID NO:
143; [0352] (14A-e) a protein comprising an amino acid sequence
encoded by a nucleic acid that hybridizes under stringent
conditions with a nucleic acid having a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 143; [0353]
(14B) a protein comprising at least one amino acid sequence
selected from the group consisting of SEQ ID NOs: 144-149,
preferably a protein comprising at least 2, 3, 4, 5 or all
sequences of the amino acid sequences. As referred to above, the
amino acid sequence of any of SEQ ID NOs: 144-149 may be an amino
acid sequence derived therefrom by deletion, substitution,
insertion or addition of one or several amino acids.
[0354] The proteins of (14A-a) to (14A-e) and (14B) as defined
above also include those proteins whose amino acid residues are
modified by phosphorylation, sugar chain modification,
aminoacylation, ring-opening, deamination or the like.
[0355] The proteins of (14A-a) to (14A-e) and (14B) as defined
above can be proteins that are found in a protein spot with a
molecular weight of around 20 to 50 kDa, preferably around 30 to 50
kDa, more preferably around 30 to 40 kDa and an isoelectric point
of 5.0 to 9.0, preferably an isoelectric point of 6.0 to 8.0, more
preferably an isoelectric point of 6.5 to 7.5 on gels used in the
two-dimensional electrophoresis performed under the conditions
described above in the subsection titled "Identification of
antigens".
[0356] Preferably, a protein that is an antigen in spot 14 is an
antigen of an allergy to fish.
[0357] By stating herein "deletion, substitution, insertion or
addition of one or several amino acids" in relation to amino acid
sequence, it is meant that in an amino acid sequence of interest,
one or several amino acids (e.g., 30%, preferably 25%, 20%, 15%,
10%, 5%, 3%, 2%, or 1% of amino acids with respect to the total
length of the amino acid sequence) are deleted, one or several
amino acids are substituted by any other amino acids, any other
amino acids are inserted, and/or any other amino acids are
added.
[0358] Among the aforementioned modifications, substitution is
preferably conservative substitution. The "conservative
substitution" refers to the substitution of a certain amino acid
residue by a different amino acid residue having similar
physicochemical characteristics, and can be any type of
substitution as long as it does not substantially change the
characteristics of the structure of the original sequence for
example, any type of substitution is acceptable as long as any
substituted amino acids do not disrupt the helical structure of the
original sequence or other secondary structures that characterize
the original sequence. The following gives examples of separate
groups of amino acid residues that are conservatively substitutable
with each other, but substitutable amino acid residues are not
limited to the examples given below. [0359] Group A: leucine,
isoleucine, valine, alanine, methionine [0360] Group B: aspartic
acid, glutamic acid [0361] Group C: asparagine, glutamine [0362]
Group D: lysine, arginine [0363] Group E: serine, threonine [0364]
Group F: phenylalanine, tyrosine
[0365] In the case of non-conservative substitution, one member
belonging to one of the aforementioned groups can be replaced with
a member belong to any other group. For example, in order to
eliminate the possibility of unwanted sugar-chain modification,
amino acids of group B, D or E as listed above may be substituted
by those of any other group. Also, cysteines may be deleted or
substituted by any other amino acids to prevent them from being
folded into a protein in its tertiary structure. Also, in order to
maintain the balance between hydrophilicity and hydrophobicity or
to increase hydrophilicity for the purpose of facilitating
synthesis, any amino acids may be substituted in consideration of
the hydropathy scales of amino acids, which are a measure of the
hydrophilic and hydrophobic properties of amino acids (J. Kyte and
R. Doolittle, J. Mol. Biol., Vol.157, p.105-132, 1982).
[0366] As referred to herein, the percent identity between two
amino acid sequences can be determined by visual inspection and
mathematical calculation. Alternatively, the percent identity can
be determined using a computer program. Examples of such computer
programs include BLAST and ClustalW. In particular, various
conditions (parameters) for identity searches with the BLAST
program are described in Altschul, et al. (Nucl. Acids. Res., 25,
p.3389-3402, 1997), and are publicly available on the websites of
the National Center for Biotechnology Information (NCBI) and DNA
Data Bank of Japan (DDBJ) (Altschul, et al., BLAST Manual,
Altschul, et al., NCB/NLM/NIH Bethesda, Md. 20894). Also, the
percent identity can be determined using a genetic information
processing software program, such as GENETYX Ver.7 (Genetyx
Corporation), DINASIS Pro (Hitachi Software Engineering Co., Ltd.),
or Vector NTI (Infomax Inc.).
[0367] By stating herein "deletion, substitution, insertion or
addition of one or several nucleotides" in relation to nucleotide
sequence, it is meant that in a nucleotide sequence of interest,
one or several nucleotides (e.g., 30%, preferably 25%, 20%, 15%,
10%, 5%, 3%, 2% or 1%, of nucleotides with respect to the total
length of the nucleotide sequence) are deleted, one or several
nucleotides are substituted by any other nucleotides, any other
nucleotides are inserted, and/or any other nucleotides are added.
It is preferable that such a nucleotide deletion, substitution,
insertion or addition should not give rise to a frame shift in an
amino acid coding sequence.
[0368] As referred to herein, the percent identity between two
nucleotide sequences can be determined by visual inspection and
mathematical calculation. Alternatively, the percent identity can
be determined using a computer program. Examples of such sequence
comparison computer programs include the BLASTN program, version
2.2.7, available on the website of the National Library of Medicine
(http://www.ncbi.nlm.nih.gov/blast/b12seq/b1s.html) (Altschul, et
al. (1990) J. Mol. Biol., 215: 403-10), or the WU-BLAST 2.0
algorithm. Standard default parameter settings for WU-BLAST 2.0 are
found and available on the following website:
http://blast.wustl.edu.
[0369] As referred to herein, "under stringent conditions" means
that hybridization takes place under moderately or highly stringent
conditions. To be specific, the moderately stringent conditions can
be easily determined by those having ordinary skill in the art on
the basis of, for example, the length of DNA. Basic conditions are
described in Sambrook, et al., Molecular Cloning: A Laboratory
Manual, 3rd ed., ch.6-7, Cold Spring Harbor Laboratory Press, 2001.
The moderately stringent conditions include hybridization under the
conditions of preferably 1.times.SSC to 6.times.SSC at 42.degree.
C. to 55.degree. C., more preferably 1.times.SSC to 3.times.SSC at
45.degree. C. to 50.degree. C., most preferably 2.times.SSC at
50.degree. C. In the case of using a hybridization solution
containing, for example, about 50% formamide, a temperature around
5 to 15.degree. C. lower than the foregoing should be adopted.
Washing is also carried out under the conditions of 0.5.times.SSC
to 6.times.SSC at 40.degree. C. to 60.degree. C. In the process of
hybridization and washing, generally 0.05% to 0.2% SDS, preferably
about 0.1% SDS, may be added. Likewise, the highly stringent
conditions can be easily determined by those having ordinary skill
in the art on the basis of, for example, the length of DNA.
Generally, the highly stringent (high stringent) conditions include
hybridization and/or washing at a higher temperature and/or a lower
salt concentration than those adopted under the moderately
stringent conditions. For example, hybridization is carried out
under the conditions of 0.1.times.SSC to 2.times.SSC at 55.degree.
C. to 65.degree. C., more preferably 0.1.times.SSC to 1.times.SSC
at 60.degree. C. to 65.degree. C., most preferably 0.2.times.SSC at
63.degree. C. Washing is carried out under the conditions of
0.2.times.SSC to 2.times.SSC at 50.degree. C. to 68.degree. C.,
more preferably 0.2.times.SSC at 60 to 65.degree. C.
[0370] Antigens may be obtained by separating and purifying them
from fish using a combination of protein purification methods well
known to those skilled in the art. Also, antigens may be obtained
by expressing them as recombinant proteins using a genetic
recombination technique well known to those skilled in the art and
by separating and purifying them using protein purification methods
well known to those skilled in the art.
[0371] Exemplary protein purification methods include:
solubility-based purification methods such as salt precipitation
and solvent precipitation; purification methods based on molecular
weight difference, such as dialysis, ultrafiltration, gel
filtration and SDS-PAGE; charge-based purification methods such as
ion exchange chromatography and hydroxylapatite chromatography;
specific affinity-based purification methods such as affinity
chromatography; purification methods based on hydrophobicity
difference, such as reverse-phase high-performance liquid
chromatography; and purification methods based on isoelectric point
difference, such as isoelectric focusing.
[0372] Preparation of a protein by a genetic recombination
technique is carried out by preparing an expression vector
comprising an antigen-encoding nucleic acid, introducing the
expression vector into appropriate host cells by gene transfer or
genetic transformation, culturing the host cells under suitable
conditions for expression of a recombinant protein, and recovering
the recombinant protein expressed in the host cells.
[0373] The "vector" refers to a nucleic acid that can be used to
introduce a nucleic acid attached thereto into host cells. The
"expression vector" is a vector that can induce the expression of a
protein encoded by a nucleic acid introduced thereby. Exemplary
vectors include plasmid vectors, viral vectors, and the like. Those
skilled in the art can select an appropriate expression vector for
the expression of a recombinant protein depending on the type of
host cells to be used. To facilitate purification, an affinity tags
such as 6 His residues may be included. Moreover, a protein may be
synthesized so that a signal sequence is included and the protein
is secreted out of cells.
[0374] The "host cells" refers to cells that undergo gene transfer
or genetic transformation by a vector. The host cells can be
appropriately selected by those skilled in the art depending on the
type of the vector to be used. The host cells can be derived from
prokaryotes such as Escherichia coli (E. coli). When prokaryotic
cells like E. coli are used as host cells, the antigen of the
present invention may be designed to contain an N-terminal
methionine residue in order to facilitate the expression of a
recombinant protein in the prokaryotic cells. The N-terminal
methionine can be cleaved from the recombinant protein after
expression. Also, the host cells may be cells derived from
eukaryotes, such as single-cell eukaryotes like yeast, plant cells
and animal cells (e.g., human cells, monkey cells, hamster cells,
rat cells, murine cells or insect cells) or silkworm.
[0375] Gene transfer or genetic transformation of an expression
vector into host cells can be carried out as appropriate by
following a technique known to those skilled in the art. Those
skilled in the art can make possible the expression of a
recombinant protein by selecting suitable conditions for the
expression of the recombinant protein as appropriate depending on
the type of host cells and culturing the host cells under the
selected conditions. Then, those skilled in the art can homogenize
the host cells having the expressed recombinant protein, and
separate and purify an antigen expressed as the recombinant protein
from the resulting homogenate by using an appropriate combination
of such protein purification methods as mentioned above.
[0376] Diagnostic Kit and Method
[0377] The present invention provides a method for providing an
indicator for diagnosing a fish allergy in a subject, the method
comprising the steps of: [0378] (i) contacting a sample obtained
from the subject with an antigen, wherein the sample is a solution
comprising an IgE antibody; [0379] (ii) detecting binding between
an IgE antibody present in the sample obtained from the subject and
the antigen; and [0380] (iii) when the binding between the IgE
antibody in the subject and the antigen is detected, an indicator
of the fact that the subject has a fish allergy is provided; [0381]
wherein the antigen is at least one of the proteins identified as
the aforementioned antigens (1) to (9) or at least one of the
proteins identified as the aforementioned antigens (10) to
(14).
[0382] The sample obtained from a subject is a solution containing
an IgE antibody, as collected from the subject. Examples of such
solutions include blood, saliva, sputum, snivel, urine, sweat, and
tear. The sample obtained from the subject may be subjected to
pretreatment for increasing the concentration of an IgE antibody in
the sample before being contacted with an antigen. The pretreatment
of a sample may involve, for example, collection of the serum and
the plasma from the blood. Furthermore, Fab portions that are
binding portions with the antigens may be purified. In a
particularly preferred embodiment, the step (i) mentioned above is
carried out by contacting an IgE antibody present in the serum
obtained from a subject with an antigen.
[0383] The IgE antibody may be the IgE antibody itself or mast
cells or the like to which the IgE antibody is bound.
[0384] Detection of contact and binding between the sample obtained
from a subject and an antigen can be carried out by using a known
method. Examples of such methods that can be used include detection
by ELISA (Enzyme-Linked Immunosorvent Assay), sandwich immunoassay,
immunoblotting, immunoprecipitation, or immunochromatography. These
are all techniques involving contacting and binding an IgE antibody
from a subject with an antigen and detecting the IgE antibody
specifically bound to the antigen. Examples thereof include methods
involving bringing an enzyme-labeled secondary antibody in contact,
adding a substrate (usually a chromogenic or luminescent reagent)
of the enzyme, and detecting the product of the enzymatic reaction;
methods involving bringing a biotinylated secondary antibody in
contact, adding an avidin-conjugated dye, and detecting the dye;
and methods involving detecting a fluorescently labeled secondary
antibody. Also, detection by a measurement method that permits the
evaluation of binding between an antigen and an IgE antibody, such
as surface plasmon resonance (SPR), can be used. A plurality of
antigen-specific IgE antibodies may be mixed.
[0385] The antigen may be provided as an isolated antigen in a
state immobilized on a carrier. In this case, the steps (i) and
(ii) mentioned above can be carried out using ELISA, sandwich
immunoassay, immunochromatography, surface plasmon resonance, or
the like. Also, the step (i) mentioned above is carried out by
contacting the sample obtained from a subject with an
antigen-immobilized surface. The isolated antigen may be obtained
by separating and purifying it from fish using a combination of
protein purification methods well known to those skilled in the
art, or by preparing it using a genetic recombination technique.
Moreover, the antibody that binds to the antigen may be in an
immobilized state.
[0386] The antigen may be in an unimmobilized state to a carrier.
In this case, the steps (1) and (ii) mentioned above can be carried
out by flow cytometry or the like and the presence of the antigen
bound to an antibody may be confirmed using a laser beam. Examples
include basophil activation test (BAT) in which an antigen binds to
an antibody and the amount of the surface antigen CD203c which is
expressed when basophils in the sample are activated is measured,
and the like. Another example is histamine release test (HRT) in
which it is examined whether histamine is released or not by
bringing an antigen in contact with hemocytes via the binding to an
antibody in a sample.
[0387] The antigen may be transferred from a state separated by
two-dimensional electrophoresis and detected by immunoblotting. The
two-dimensional electrophoresis is a technique for separating a
protein sample by performing isoelectric focusing in the first
dimension and performing SDS-PAGE (SDS-polyacrylamide gel
electrophoresis) in the second dimension. The conditions for
two-dimensional electrophoresis are not particularly limited as
long as the conditions permit the separation of the antigen of the
present invention. For example, the conditions for two-dimensional
electrophoresis as described above in the subsection titled
"Identification of antigens" can be adopted. Also, the
electrophoresis conditions may be determined by reference to the
descriptions in PTLs 1 to 4 mentioned above. For example,
two-dimensional electrophoresis can be carried out under the
conditions that satisfy at least one selected from the group
consisting of the following requirements: [0388] (A) the
isoelectric focusing gels used in the first dimension should have a
gel-strip length of 5 to 10 cm and a gel pH range of 3 to 10, and
the pH gradient of the gels in the direction of electrophoresis
should be as follows: where the gel-strip length up to pH 5 is
taken as "a", that length from pH 5 to 7 as "b", and that length
above pH 7 as "c", the relations "a<b" and "b>c" are
satisfied; [0389] (B) in the case of (A), when the total gel-strip
length is taken as 1, "a" should be in the range of 0.15 to 0.3,
"b" should be in the range of 0.4 to 0.7, and "c" should be in the
range of 0.15 to 0.3; [0390] (C) in the first dimensional
isoelectric focusing, a constant voltage step should be performed
by applying a constant voltage ranging from 100 V to 600 V per gel
strip containing a sample, and after the electrophoresis variation
width during electrophoresis for 30 minutes falls within the range
of 5 .mu.A, a voltage-increasing step should be started at which
the voltage is increased from the aforementioned constant voltage;
[0391] (D) in the case of (C), the final voltage at the
voltage-increasing step should be in the range of 3000 V to 6000 V;
[0392] (E) the isoelectric focusing gels used in the first
dimension should have a longitudinal gel-strip length of 5 to 10
cm, and the electrophoresis gels used in the second dimension
should have a gel concentration at the distal end in the direction
of electrophoresis, which is in the range of 3 to 6%; and [0393]
(F) in the case of (E), the electrophoresis gels used in the second
dimension should have a gel concentration at the proximal end in
the direction of electrophoresis, which is set to a higher value
than that at the distal end in the direction of
electrophoresis.
[0394] The aforementioned antigens (1) to (9) and the
aforementioned antigens (10) to (14) are antigens which
specifically bind to IgE antibodies in patients with an allergy to
fish. Therefore, when binding between an IgE antibody from a
subject and the antigen is detected, an indicator of the fact that
the subject has a fish allergy is provided.
[0395] The present invention further provides a kit for diagnosing
an allergy to a fish, comprising at least one of the aforementioned
antigens (1) to (9) or at least one of the aforementioned antigens
(10) to (14). The diagnostic kit of this invention may be used in
the aforementioned method for providing an indicator for diagnosing
an allergy to a fish or in a diagnostic method as described later.
The diagnostic kit of this invention comprises at least one of the
aforementioned antigens (1) to (9) or at least one of the
aforementioned antigens (10) to (14) and may comprise a chromogenic
substrate or luminescent substrate serving as a substrate of an
enzyme-labeled anti-IgE antibody and the enzyme or a biotinylated
anti-IgE antibody and an avidin-conjugated dye that binds to the
biotin. Also, a fluorescent-labeled anti-IgE antibody may be used.
In the diagnostic kit of this invention, the antigen may be
provided in a state immobilized on a carrier. The diagnostic kit of
this invention may also be provided together with instructions on
the procedure for diagnosis or a package containing said
instructions.
[0396] In another embodiment, the diagnostic kit comprises a
companion diagnostic agent for an allergy to fish. The companion
diagnostic agent is used to analyze the reactivity of a
pharmaceutical product for the purpose of identifying a patient in
which the pharmaceutical product is expected to work or a patient
at risk for serious side effects from the pharmaceutical product,
or optimizing a therapy using the pharmaceutical product. Here,
examples of optimizing a therapy include determination of the
dosage and administration, decision of stopping the administration,
determination of the allergen component to be used for the
induction of immune tolerance, and the like.
[0397] The present invention further provides a composition for
diagnosing an allergy to a fish, comprising at least one of the
aforementioned antigens (1) to (9) or at least one of the
aforementioned antigens (10) to (14). The diagnostic composition of
this invention can be used in a diagnostic method as described
below. The diagnostic composition of this invention may further
comprise a pharmaceutically acceptable carrier and/or additives
commonly used with the antigen of this invention depending on the
need.
[0398] In one embodiment, the present invention provides a method
for diagnosing an allergy to a fish in a subject, the method
comprising: [0399] (i) contacting a sample obtained from the
subject with an antigen; [0400] (ii) detecting binding between an
IgE antibody present in the sample obtained from the subject and
the antigen; and [0401] (iii) when the binding between the IgE
antibody in the subject and the antigen is detected, diagnosing the
subject as being allergic to a fish; [0402] wherein the antigen is
at least one of the proteins identified as the aforementioned
antigens (1) to (9) or at least one of the proteins identified as
the aforementioned antigens (10) to (14). In this method, each of
the steps (i) and (ii) is performed as described above regarding
each of the corresponding steps of the method for providing an
indicator for diagnosing an allergy to a fish.
[0403] In another embodiment, the present invention provides a
method for diagnosing an allergy to a fish in a subject, the method
comprising administering to the subject at least one of the
aforementioned antigens (1) to (9) or at least one of the
aforementioned antigens (10) to (14). This method may be performed
in the form of a skin test characterized by applying the antigen
onto the skin. Examples of the skin test include various forms of
tests, such as: a prick test in which a diagnostic composition is
applied onto the skin and then a tiny prick to such an extent as
not to provoke bleeding is made in the skin to allow an antigen to
infiltrate into the skin, thereby observing a skin reaction; a
scratch test in which a diagnostic composition is applied onto the
skin and then the skin is lightly scratched to observe a reaction;
a patch test in which a diagnostic composition in the form of
cream, ointment, etc. is applied onto the skin to observe a
reaction; and an intracutaneous test in which an antigen is
administered intracutaneously to observe a reaction. The method for
infiltrating a diagnostic composition into the skin in a prick test
or a scratch test may be a method involving bringing a tip of a
lancet in contact with a diagnostic composition and wounding the
skin by pricking the skin with the contact site to infiltrate the
composition. If a skin reaction such as swelling occurs in a skin
portion to which the antigen has been applied, the subject of
interest is diagnosed as having an allergy to a fish. The amount of
the antigen to be applied to the skin in such tests can be, for
example, not more than 100 .mu.g per dose.
[0404] In the allergic diagnosis, a load test for the purpose of
identifying the antigen is often performed. At least one of the
aforementioned antigens (1) to (9) or at least one of the
aforementioned antigens (10) to (14) may be used as an active
ingredient of a load test for diagnosing an allergy to fish. Here,
the antigen protein to be used in the load test may be a protein
that has been expressed and purified and may be a protein that has
been expressed in a food and a food material, for example,
rice-based vaccine produced by transforming rice with a gene of a
cedar pollen antigen to express the antigen protein therein.
[0405] In yet another embodiment, the present invention provides at
least one of the aforementioned antigens (1) to (9) or at least one
of the aforementioned antigens (10) to (14) for use in the
diagnosis of an allergy to a fish. Here, the present invention
encompasses the provision of at least one of the aforementioned
antigens (1) to (9) or (10) to (14) mixed with a known antigen.
[0406] In still another embodiment, the present invention provides
a use of at least one of the aforementioned antigens (1) to (9) or
at least one of the aforementioned antigens (10) to (14) for the
production of a diagnostic agent for an allergy to a fish.
[0407] Pharmaceutical Composition and Treatment Method
[0408] The present invention provides a pharmaceutical composition
comprising at least one of the aforementioned antigens (1) to (9)
or at least one of the aforementioned antigens (10) to (14).
[0409] In one embodiment, the aforementioned pharmaceutical
composition is used for the treatment of an allergy to fish.
[0410] The present invention also provides a method for treating an
allergy to fish, the method comprising administering at least one
of the aforementioned antigens (1) to (9) or at least one of the
aforementioned antigens (10) to (14) to a patient in need of a
treatment for an allergy to a fish.
[0411] In another embodiment, the present invention provides at
least one of the aforementioned antigens (1) to (9) or at least one
of the aforementioned antigens (10) to (14) for use in the
treatment of an allergy to fish. In yet another embodiment, the
present invention provides use of at least one of the
aforementioned antigens (1) to (9) or at least one of the
aforementioned antigens (10) to (14) for the production of a
therapeutic agent for an allergy to fish.
[0412] In the process of allergy treatment, a hyposensitization
therapy aiming at inducing immunological tolerance by administering
an antigen to a patient is often adopted. At least one of the
aforementioned antigens (1) to (9) or at least one of the
aforementioned antigens (10) to (14) can be used as an active
ingredient for a hyposensitization therapy for allergy to fish.
Here, the antigen protein to be used in the hyposensitization
therapy may be a protein that has been expressed and purified and
may be a protein that has been expressed in a food and a food
material, for example, rice-based vaccine produced by transforming
rice with a gene of a cedar pollen antigen to express the antigen
protein therein.
[0413] The pharmaceutical composition of the present invention can
be administered by common administration routes. Examples of common
administration routes include oral, sublingual, percutaneous,
intracutaneous, subcutaneous, intravascular, intranasal,
intramuscular, intraperitoneal, and intrarectal
administrations.
[0414] The pharmaceutical composition of the present invention can
be used as a pharmaceutical composition to which a commonly used
pharmaceutically acceptable adjuvant or excipient or any other
additives (e.g., stabilizer, solubilizer, emulsifier, buffer,
preservative, colorant) are added by a conventional method together
with the antigen of this invention depending on the need. The
dosage form of the pharmaceutical composition can be selected by
those skilled in the art as appropriate depending on the
administration route. The pharmaceutical composition can be in the
form of, for example, tablet, capsule, troche, sublingual tablet,
parenteral injection, intranasal spray, poultice, solution, cream,
lotion, or suppository. The administration dose, frequency and/or
period of the pharmaceutical composition of this invention can be
selected by a physician as appropriate depending on the
administration route and the patient's condition and
characteristics such as age and body weight. For example, the
pharmaceutical composition may be administered to an adult patient
at a dose of not more than 100 .mu.g per dose. The administration
interval can be, for example, once daily, once weekly, twice
monthly, once per three months or so. The administration period can
be, for example, several weeks to several years. The pharmaceutical
composition may be administered in such a manner that the dose is
increased in incremental steps over the administration period.
[0415] Tester
[0416] The present invention provides a tester comprising an
antibody for at least one of the aforementioned antigens (1) to (9)
or at least one of the aforementioned antigens (10) to (14).
[0417] The antibody can be prepared by a conventional method: For
example, the antibody may be prepared by immunizing a mammal such
as rabbit with one of the aforementioned antigens (1) to (9) or one
of the aforementioned antigens (10) to (14). The antibody may be an
IgE antibody, a polyclonal antibody, a monoclonal antibody, or an
antigen-binding fragment thereof (e.g., Fab, F(ab').sub.2,
Fab').
[0418] Further, in the aforementioned tester, the antibody may be
provided in a form bound to a carrier. The type of the carrier is
not particularly limited as long as it is usable for detection of
binding between an antibody and an antigen. Any given carrier known
to those skilled in the art can be used.
[0419] Examples of a method for determining the presence or absence
of an antigen include: [0420] A method in which a prepared tester
comprising an IgE antibody is contacted with a sample obtained from
a food or food material, and ELISA or the like method is, for
example, used to detect whether there is a binding between the IgE
antibody and an antigen in the sample, and if the binding between
the IgE antibody and the antigen is detected, it is determined that
the antibody remains in the food or food material, of interest.
[0421] A method in which proteins are extracted from a food or food
material, and the proteins are subjected to electrophoresis, and it
is detected with an antibody whether there is a spot band of the
antigen. [0422] A method in which a food or food material is
infiltrated into a filter paper or the like and an antibody
solution is reacted with the filter paper to detect an antigen
present in the food.
[0423] The present invention encompasses, in another embodiment, a
tester for determining the presence or absence of an antigen for an
allergy to fish in an object of interest, the tester comprising a
primer having a nucleotide sequence complementary to at least one
part of one of the nucleotide sequences set forth in SEQ ID NO: 1,
4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, or 143. Without
limiting, the primer has a nucleotide sequence complementary to
preferably 12 residues, 15 nucleotides, 20 nucleotides, or 25
nucleotides in the sequence of a 3' terminal or middle portion of
at least one part of one sequence of the nucleotide sequences set
forth in, for example, SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60,
69, 109, 120, 137, or 143. Particularly when mRNA is targeted, the
tester comprises a poly A tail-complementarity primer. In a
preferred embodiment, the tester comprising the primer may further
comprise a primer comprising a nucleotide sequence of a 5' terminal
portion of at least one sequence of the nucleotide sequences set
forth in SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120,
137, or 143, preferably a nucleotide sequence consisting of 12
nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides.
[0424] For example, the presence or absence of the antigen is
determined by amplifying DNA or cDNA by polymerase chain reaction
(PCR) including RT-PCR by using the aforementioned primer with DNA
or mRNA obtained from fish as a template and comparing the
amplified DNA or cDNA sequence(s) with SEQ ID NO: 1, 4, 9, 18, 25,
32, 42, 55, 60, 69, 109, 120, 137, or 143. Examples of the method
of the amplification of mRNA by PCR include RACE and the like. In
the determination, the antigen is determined to be present when the
comparison between the amplified DNA or cDNA and SEQ ID NO: 1, 4,
9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, or 143 indicates the
presence of point mutation(s) encoding the same amino acid(s), or
when the amino acid sequence encoded by the amplified DNA or cDNA
has 70% or more, preferably 80, 90, 95, 98, or 99% or more identity
with an amino acid sequence of SEQ ID NO: 2, 5, 10, 19, 26, 33, 43,
56, 61, 70, 110, 121, 138, or 144, even if the nucleotide sequence
of the amplified DNA or cDNA is modified from any of the nucleotide
sequences of SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109,
120, 137, or 143 by insertion, deletion, substitution, or addition
of nucleotide(s).
[0425] In one embodiment, the aforementioned tester is used to
determine the presence or absence of an antigen in food materials
(fish or fish eggs) or in products of interest in a food production
line. The tester may also be used for quality inspection of
production lines and pre-shipment products by manufacturers, or may
be used for self-checking of the presence or absence of an antigen
in a food or food material of interest by consumers.
[0426] Allergen-Free Food and the Like
[0427] The present invention provides fish or fish eggs in which at
least one of the aforementioned antigens (1) to (9) or at least one
of the aforementioned antigens (10) to (14) is eliminated or
reduced, a processed product of the fish or the fish eggs or fish
which lay the fish eggs or have hatched from the eggs.
[0428] The method for eliminating or reducing the antigen of the
present invention in fish, fish eggs, the processed product of the
fish or the fish eggs or the fish which lay the fish eggs or have
hatched from the eggs is not limited. The elimination or reduction
of the antigen of the present invention may be conducted by any
method, as long as the method permits the elimination or reduction
of the antigen.
[0429] For example, the fish or fish eggs in which the antigen of
the present invention is eliminated or reduced may be obtained by
preparing fish or fish eggs in which the expression of the antigen
of the present invention is knocked out, using a gene knock-out
technique.
[0430] As the gene knock-out technique, there can be used any
methods known to those skilled in the art. For example, Oishi, et
al. (Scientific Reports, Vol.6, Article number: 23980, 2016,
doi:10.1038/srep23980) describes that the genome editing technique
CRISPER/Cas9 is applied to primordial germ cells to obtain
individual animals deficient in an allergen gene. The fish or fish
eggs devoid of the antigen of this invention may also be obtained
by using the same technique. Moreover, fish or fish eggs in which
the antigen of the present invention is eliminated or reduced may
be obtained by mating by artificial insemination with fish or fish
eggs that do not contain the antigen or contains a low content of
the antigen. The artificial mating of fish or fish eggs can be
performed by a conventional method.
[0431] The processed product of the fish or fish eggs in which the
antigen of the present invention is eliminated or reduced may be a
processed product made from fish in which the antigen of the
present invention is eliminated or reduced. In the case of using
ordinary fish or fish eggs as a source ingredient, a treatment for
removing or reducing the antigen of the present invention is
performed before or after preparation of a processed product of
fish or fish eggs. Examples of a method of eliminating or reducing
the antigen of the present invention in a processed product of fish
or fish eggs made from ordinary fish or fish eggs include methods
for eliminating the protein component in a food or a food material,
such as elution with a high pressure treatment and a neutral salt
solution and hot steam and methods for hydrolysis, denaturation, or
amino acid modification (chemical modification or elimination of
side chains) by heat treatment and acid treatment. The fish in
which the antigen of the present invention is eliminated or reduced
may be fish that have hatched and grown from the aforementioned
fish eggs in which the antigen of the present invention is
eliminated or reduced. Moreover, the fish eggs in which the antigen
of the present invention is eliminated or reduced may be those
obtained from the aforementioned fish in which the antigen of the
present invention is eliminated or reduced.
EXAMPLES
[0432] The following describes examples of the present invention.
The technical scope of this invention is not limited by these
examples.
Example 1: Confirmation of a Protein Pattern
[0433] Proteins contained in salmon were investigated using a
two-dimensional electrophoresis method described below.
[0434] Protein Extraction
[0435] The extraction and the purification of proteins contained in
salmon were performed as follows. A solubilization agent (Mammalian
Lysis Buffer (MCLI), SIGMA) was added to flesh of salmon and
proteins were extracted, and then a urea buffer was added to obtain
a protein extract. The composition of the urea buffer is as
follows.
[0436] 30 mM Tris
[0437] 2 M Thiourea
[0438] 7 M Urea
[0439] 4% (w/v) CHAPS:
[0440] 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate
[0441] Moderate amount of dilute hydrochloric acid
[0442] Distilled water was added and the total volume was adjusted
to 100 mL. pH was 8.5.
[0443] Subsequently, 25 .mu.g each in terms of protein weight was
mixed to obtain an extract.
[0444] Thereafter, the precipitation procedure was repeated twice
using a 2D-CleanUP Kit (produced by GE). In the first round of
precipitation, the collected liquid protein extract was
precipitated by adding TCA (trichloroacetic acid) thereto and the
precipitated product produced by this procedure (TCA-precipitated
product) was collected. In the second round of precipitation, the
TCA-precipitated product collected above was further precipitated
by adding acetone thereto and the precipitated product (sample)
produced by this procedure was collected.
[0445] Preparation of a Sample Solution
[0446] Part of the collected sample (40 .mu.g on a protein weight
basis) was dissolved in 150 .mu.L of a DeStreak Rehydration
Solution (produced by GE), which is a swelling buffer for
first-dimensional isoelectric focusing gels, thereby obtaining a
sample solution for first-dimensional isoelectric focusing (sample
solution for swelling). The constituents of the DeStreak
Rehydration Solution are as mentioned below.
[0447] 7M thiourea
[0448] 2M urea
[0449] 4% (w/v) of CHAPS
[0450] 0.5% (v/v) IPG buffer; produced by GE
[0451] Moderate amount of BPB (bromophenol blue)
[0452] Impregnation of the Sample into First-Dimensional
Isoelectric Focusing Gels
[0453] First-dimensional isoelectric focusing gel strips
(Immobiline Drystrip IPG gels (pH 3-10NL); produced by GE) were
immersed in 140 .mu.L of the foregoing sample solution for
first-dimensional isoelectric focusing (sample solution for
swelling) and impregnated with the solution at room temperature
overnight.
[0454] In this example, an IPGphor electrophoresis system produced
by GE was used.
[0455] An electrophoresis tray was filled with silicone oil. Filter
paper moisten with water was positioned at both ends of the gel
strips impregnated with the sample, and the gel strips were set in
the electrophoresis tray such that the gel strips were covered with
silicone oil. Electrodes were placed on the gel strips with the
filter paper intervening therebetween.
[0456] The maximum current of the isoelectric focusing system was
set to 75 .mu.A per gel strip, and the first-dimensional
isoelectric focusing was carried out according to the following
voltage program: (1) a constant voltage step was performed at a
constant voltage of 300 V until the volt-hours reached 750 Vhr (the
current variation width during electrophoresis for 30 minutes
before the end of this step was 5 .mu.A); (2) the voltage was
increased gradually to 1000 V for 300 Vhr; (3) the voltage was
further increased gradually to 5000 V for 4500 Vhr; and then (4)
the voltage was held at a constant voltage of 5000 V until the
total Vhr reached 12000.
[0457] SDS Equilibration of Isoelectric Focusing Gels
[0458] After the aforementioned first-dimensional isoelectric
focusing was done, the gel strips were taken out of the isoelectric
focusing system, immersed in an equilibration buffer containing a
reducing agent, and shaken at room temperature for 15 minutes. The
constituents of the equilibration buffer containing the reducing
agent are as mentioned below.
[0459] 100 mM Tris-HCl (pH 8.0)
[0460] 6M urea
[0461] 30% (v/v) glycerol
[0462] 2% (w/v) SDS
[0463] 1% (w/v) DTT
[0464] Next, the equilibration buffer containing the reducing agent
was removed, and then the gel strips were immersed in an
equilibration buffer containing an alkylating agent and shaken at
room temperature for 15 minutes to obtain SDS-equilibrated gels.
The constituents of the equilibration buffer containing the
alkylating agent are as mentioned below.
[0465] 100 mM Tris-HCl (pH 8.0)
[0466] 6M urea
[0467] 30% (v/v) glycerol
[0468] 2% (w/v) SDS
[0469] 2.5% (w/v) iodoacetamide
[0470] Second-Dimensional SDS-PAGE
[0471] In this example, the XCell SureLock Mini-Cell
electrophoresis system produced by Life Technologies was used. The
second-dimensional electrophoresis gels used were NuPAGE 4-12%
Bis-Tris Gels produced by Life Technologies. Also, an
electrophoresis buffer composed of the following constituents was
prepared and used.
[0472] 50 mM MOPS
[0473] 50 mM Tris base
[0474] 0.1% (w/v) SDS
[0475] 1 mM EDTA
[0476] Further, an agarose solution for gel adhesion was used in
this example, which was prepared by dissolving 0.5% (w/v) Agarose S
(produced by Nippon Gene Co., Ltd.) and a moderate amount of BPB
(bromophenol blue) in the electrophoresis buffer.
[0477] SDS-PAGE wells were washed well with the electrophoresis
buffer, and then the buffer used for the washing was removed. Next,
the washed wells were charged with the fully dissolved agarose
solution for gel adhesion. Next, the SDS-equilibrated gel strips
were immersed in agarose and closely adhered to second-dimensional
electrophoresis gels using tweezers. After it was confirmed that
agarose was fully fixed with the gels being closely adhered to each
other, electrophoresis was performed at a constant voltage of 200 V
for about 45 minutes.
[0478] Fluorescent Staining of Gels
[0479] The gels were fluorescently stained with SYPRO Ruby
(produced by Life Technologies).
[0480] First, an airtight container to be used was washed well in
advance with 98% (v/v) ethanol. The electrophoresed
second-dimensional electrophoresis gel strips were taken out of the
SDS-PAGE system, placed onto the washed airtight container, and
treated twice by immersion in 50% (v/v) methanol and 7% (v/v)
aqueous solution containing acetic acid for 30 minutes. Then, a
further immersion treatment was done for 10 minutes, with the
solution being replaced by water. Next, the second-dimensional
electrophoresis gel strips were immersed in 40 mL of SYPRO Ruby and
shaken at room temperature overnight. Thereafter, the SYPRO Ruby
was removed, and then the second dimensional electrophoresis gel
strips were washed with water and shaken in 10% (v/v) methanol and
7% (v/v) aqueous solution containing acetic acid for 30 minutes.
Further shaking was done for at least 30 minutes, with the solution
being replaced by water.
[0481] Analysis
[0482] The second-dimensional electrophoresis gels obtained through
the foregoing series of treatments were subjected to fluorescent
image scanning on Typhoon 9500 (produced by GE). The left panel of
FIG. 1A illustrates the result of two-dimensional electrophoresis
of proteins contained in flesh of salmon. Molecular weight marker
bands are found at the left of the panels. The positions of the
bands indicate particular molecular weights (KDa).
Example 2: Identification of Antigens by Immunoblotting (1)
[0483] Identification of antigens by immunoblotting was carried out
by taking all the steps up to the step of "Second-dimensional
SDS-PAGE" as described above in Example 1, followed by the steps of
"Transfer to membrane", "Immunoblotting" and "Analysis" as
described below.
[0484] Transfer to Membrane
[0485] Transfer to membrane was done using the following transfer
system and transfer buffer. [0486] Transfer system: XCell SureLock
Mini-Cell and XCell II Blot Module (produced by Life Technologies)
[0487] Transfer buffer: NuPAGE Transfer Buffer (.times.20)
(produced by Life Technologies), used in a form diluted 20-fold
with milliQ water.
[0488] To be specific, proteins in the second-dimensional
electrophoresis gels were transferred to a membrane (PVDF membrane)
according to the following procedure. [0489] (1) The PVDF membrane
was immersed in 100% methanol followed by milliQ water, and then
moved into the transfer buffer to hydrophilize the PVDF membrane.
[0490] (2) After sponge, filter paper, the second-dimensional
electrophoresis gels prepared by second-dimensional SDS-PAGE, the
hydrophilized PVDF membrane, filter paper, and sponge were put in
place in this order, the transfer system was energized at a
constant voltage of 30 V for one hour.
[0491] Immunoblotting
[0492] Immunoblotting of the membrane was carried out using, as a
primary antibody, a serum sample from a patient 1 with a fish
allergy or a serum sample from a non-fish-allergic subject. This
Fish-allergic patient 1 is a patient diagnosed as immediate-type
allergy to fish. This patient developed allergic symptoms to
salmon, horse mackerel, sea eel, black porgy, mackerel, sea bream,
cod, and young yellowtail and was positive in a prick test.
[0493] Immunoblotting of the membrane was carried out according to
the following procedure. [0494] (1) The transferred membrane was
shaken in a 5% skim milk/PBST solution (a PBS buffer containing
0.1% Tween 20 nonionic surfactant) at room temperature for one
hour. [0495] (2) The membrane was left to stand in a solution of 5%
serum as a primary antibody in 5% skim milk/PBST at room
temperature for one hour. [0496] (3) The membrane was washed with a
PBST solution (5 min..times.3 times). [0497] (4) The membrane was
left to stand in a solution of 1:5000 dilution of the secondary
antibody, anti-human IgE-HRP (horseradish peroxidase), with a 5%
skim milk/PBST solution at room temperature for one hour. [0498]
(5) The membrane was washed with a PBST solution (5 min..times.3
times). [0499] (6) The membrane was left to stand in Pierce Western
Blotting Substrate Plus (produced by Thermo Fisher Scientific) for
5 minutes.
[0500] Analysis
[0501] The membrane obtained through the foregoing series of
treatments was subjected to fluorescent image scanning on Typhoon
9500 (produced by GE).
[0502] The immunoblot obtained with the serum from Fish-allergic
patient 1 was compared with that obtained with the control serum
from Non-fish-allergic subject. Nine spots that are different from
those with the serum of Non-fish-allergic subject (FIG. 3) and
different from the known salmon allergen proteins were detected on
an immunoblot of the proteins contained in flesh of salmon with the
serum of Fish-allergic patient 1 (FIG. 1A, right panel). The
detected spots are indicated in the immunoblot (FIG. 1A, right
panel).
[0503] The molecular weights and isoelectric points of the 9 spots
are as follows. [0504] Spot 1: Molecular weight 90 to 120 kDa, pI
3.0 to 6.0 [0505] Spot 2: Molecular weight 120 to 160 kDa, pI 4.0
to 7.0 [0506] Spot 3: Molecular weight 90 to 120 kDa, pI 5.0 to 8.0
[0507] Spot 4: Molecular weight 90 to 120 kDa, pI 6.0 to 8.0 [0508]
Spot 5: Molecular weight 60 to 90 kDa, pI 3.0 to 6.0 [0509] Spot 6:
Molecular weight 70 to 100 kDa, pI 5.0 to 7.0 [0510] Spot 7:
Molecular weight 40 to 70 kDa, pI 4.0 to 7.0 [0511] Spot 8:
Molecular weight 40 to 70 kDa, pI 4.0 to 7.0 [0512] Spot 9:
Molecular weight 20 to 50 kDa, pI 4.0 to7.0
Example 3: Mass Spectrometry and Identification of Antigens (1)
[0513] The amino acid sequences of the antigens that form the above
nine protein spots were identified by mass spectroscopy.
[0514] To be specific, protein extraction and mass spectroscopy
were done by the following procedure. [0515] (1) Flesh of salmon
was subjected to protein extraction, two-dimensional
electrophoresis and transfer to membrane by following the
procedures described in Example 1 and 2, and the resulting membrane
was stained by shaking in a solution of 0.008% Direct blue in 40%
ethanol and 10% acetic acid. [0516] (2) Then, the membrane was
decolorized by repeating a 5-minute treatment with 40% ethanol and
10% acetic acid three times, washed with water for 5 minutes, and
then dried by air. [0517] (3) A protein spot of interest was cut
out with a clean cutter blade and put into a centrifugal tube. The
cut membrane was subjected to hydrophilization with 50 .mu.L of
methanol, followed by washing with 100 .mu.L of water twice and
then centrifugal cleaning. Thereafter, 20 .mu.L of 20 mM
NH.sub.4HCO.sub.3 and 50% acetonitrile were added. [0518] (4) 1
.mu.L of 1 pmol/.mu.L lysyl endopeptidase (produced by WAKO) was
added, and the solution was left to stand at 37.degree. C. for 60
minutes and then collected in a new centrifugal tube. After 20
.mu.L of 20 mM NH.sub.4HCO.sub.3 and 70% acetonitrile were added to
the membrane, the membrane was immersed therein at room temperature
for 10 minutes, and the resulting solution was further collected.
The solution was dissolved with 0.1% formic acid and 10 .mu.L of 4%
acetonitrile and transferred to a tube. [0519] (5) The collected
solution was dried under reduced pressure, dissolved with 15 .mu.L
of solution A (a 0.1% formic acid/4% acetonitrile solution), and
analyzed by mass spectroscopy (ESI-TOF6600, produced by AB Sciex).
[0520] (6) Identification of proteins based on the MS data obtained
with the mass spectrometer was done by searching NCBI or
UniProt.
[0521] Results
[0522] The mass spectrometry of the spots resulted in the detection
of the following amino acid sequences. [0523] Spot 1: the amino
acid sequences set forth in SEQ ID NO: 3 [0524] Spot 2: the amino
acid sequences set forth in SEQ ID NO: 6 to 8 [0525] Spot 3: the
amino acid sequences set forth in SEQ ID NO: 11 to 17 [0526] Spot
4: the amino acid sequences set forth in SEQ ID NO: 20 to 24 [0527]
Spot 5: the amino acid sequences set forth in SEQ ID NO: 27 to 31
[0528] Spot 6: the amino acid sequences set forth in SEQ ID NO: 34
to 41 [0529] Spot 7: the amino acid sequences set forth in SEQ ID
NO: 44 to 54 [0530] Spot 8: the amino acid sequences set forth in
SEQ ID NO: 57 to 59 [0531] Spot 9: the amino acid sequences set
forth in SEQ ID NO: 62 to 68
[0532] Furthermore, the spots were identified as the following
proteins by the analysis of the MS data obtained from the mass
spectrometer for spot 1 at NCBI and for spots 2 to 9 with UniProt.
[0533] Spot 1: Alpha-actinin-3 (NCBI protein accession number
XP_014051545.1, DNA accession number XM_014196070.1) (amino acid
sequence: SEQ ID NO: 2, encoding nucleotide sequence: SEQ ID NO: 1)
[0534] Spot 2: EEF1A2 binding protein-like (UniProt protein
accession number B5RI29, DNA accession number ACH85270.1) (amino
acid sequence: SEQ ID NO: 5, encoding nucleotide sequence: SEQ ID
NO: 4) [0535] Spot 3: Alpha-1,4-glucan phosphorylase (UniProt
protein accession number B5DG55, DNA accession number ACH70729.1)
(amino acid sequence: SEQ ID NO: 10, encoding nucleotide sequence:
SEQ ID NO: 9) [0536] Spot 4: Elongation factor 2 (UniProt protein
accession number C0H9N2, DNA accession number ACN10751.1) (amino
acid sequence: SEQ ID NO: 19, encoding nucleotide sequence: SEQ ID
NO: 18) [0537] Spot 5: Heat shock cognate 70 kDa protein (UniProt
protein accession number B5X3U6, DNA accession number ACI33977.1)
(amino acid sequence: SEQ ID NO: 26, encoding nucleotide sequence:
SEQ ID NO: 25) [0538] Spot 6: Serotransferrin (UniProt protein
accession number P79815, DNA accession number BAA13759.1) (amino
acid sequence: SEQ ID NO: 33, encoding nucleotide sequence: SEQ ID
NO: 32) [0539] Spot 7: Myosin binding protein H-like (UniProt
protein accession number B5DG45, DNA accession number ACH70719.1)
(amino acid sequence: SEQ ID NO: 43, encoding nucleotide sequence:
SEQ ID NO: 42) [0540] Spot 8: Desmin (Fragment) (UniProt protein
accession number Q8UWF1, DNA accession number CAC83053.1) (amino
acid sequence: SEQ ID NO: 56, encoding nucleotide sequence: SEQ ID
NO: 55) [0541] Spot 9: Capping protein (Actin filament) muscle
Z-line beta (UniProt protein accession number B5DFX6, DNA accession
number ACH70650.1) (amino acid sequence: SEQ ID NO: 61, encoding
nucleotide sequence: SEQ ID NO: 60)
Example 4: Confirmation of Antigens in Other Fish Species (1)
[0542] Using flesh of the 9 fish species, horse mackerel, sea eel,
black porgy, mackerel, sea bream, cod, young yellowtail, eel, and
flatfish, other than salmon, antigens were confirmed by the same
procedure as the procedure described in Example 1 and Example
2.
[0543] Immunoblots obtained with the serum from Fish-allergic
patient 1 was compared with that obtained with the control serum
from Non-fish-allergic subject. Spots that are different from those
with the serum from Non-fish-allergic subject (FIG. 3) and
correspond to a part of the 9 spots obtained in salmon were
detected on immunoblots (FIG. 2) of proteins contained in flesh of
each of the 9 fish species stained with the serum from
Fish-allergic patient 1.
[0544] Spots detected in each fish species are as follows. [0545]
Horse mackerel: Spots 1, 3-5, 8, and 9 [0546] Sea eel: Spots 1, 3,
5, and 9 [0547] Black porgy: Spots 1-6, 8, and 9 [0548] Mackerel:
Spots 1, 3, 5, 8, and 9 [0549] Sea bream: Spots 1, 3-6, 8, and 9
[0550] Cod: Spots 1, 3, 5, 7-9 [0551] Young yellowtail: Spots 1,
3-5, 8, 9 [0552] Eel: Spots 1, 3, 4, 6, 9 [0553] Flatfish: Spots 1,
4, 6
Example 5: Confirmation of Antigens by Immunoblotting (2)
[0554] An immunoblot obtained with serum from a fish-allergic
patient was compared with that obtained with the control serum from
Non-fish-allergic subject in the same way as in Example 2. Three
new spots (Spots 11, 12, 14) that are different from those with the
serum from Non-fish-allergic subject (FIG. 3) and different from
the known salmon allergen proteins, other than 7 (spots 1-6, 9) of
the 9 spots detected in Example 2, were detected on an immunoblot
(FIG. 1B, left panel) of proteins contained in flesh of salmon
stained with the serum from Fish-allergic patient 2. The total 10
spots detected are indicated in the immunoblot (FIG. 1B, left
panel).
[0555] The molecular weights and isoelectric points of the 3 new
spots detected are as follows. [0556] Spot 11: Molecular weight 90
to 110, pI 6.5 to 7.0 [0557] Spot 12: Molecular weight 120 to 140,
pI 5.0 to 6.0 [0558] Spot 14: Molecular weight 30 to 40, pI 6.5 to
7.5
Example 6: Mass Spectrometry and Identification of Antigens (2)
[0559] The amino acid sequences of the antigens that form the 3
spots newly detected were identified by mass spectroscopy in the
same way as in Example 3.
[0560] The mass spectrometry of the spots resulted in the detection
of the following amino acid sequences. [0561] Spot 11: Amino acid
sequences set forth in SEQ ID NOs: 111 to 119 [0562] Spot 12: Amino
acid sequences set forth in SEQ ID NOs: 122 to 136 [0563] Spot 14:
Amino acid sequences set forth in SEQ ID NOs: 145 to 149
[0564] Furthermore, the spots were identified as the following
proteins by the analysis of the MS data obtained from the mass
spectrometer for the spots at NCBI. [0565] Spot 11: Glycogen
phosphorylase, muscle form-like (NCBI protein accession number
XP_013984904.1, DNA accession number XM_014129429.1) (amino acid
sequence: SEQ ID NO: 110, encoding nucleotide sequence: SEQ ID NO:
109) [0566] Spot 12: Myosin-binding protein C, fast-type-like (NCBI
protein accession number XP_014014310.1, DNA accession number
XM_014158835.1) (amino acid sequence: SEQ ID NO: 121, encoding
nucleotide sequence: SEQ ID NO: 120) [0567] Spot 14: L-lactate
dehydrogenase A chain-like (NCBI protein accession number
XP_014003141.1, DNA accession number XM_014147666.1) (amino acid
sequence: SEQ ID NO: 144, encoding nucleotide sequence: SEQ ID NO:
143)
Example 7: Confirmation of Antigens by Immunoblotting (3)
[0568] An immunoblot obtained with the serum from a fish-allergic
patient was compared with that obtained with the control serum from
Non-fish-allergic subject, in the same way as in Example 2. Two new
spots (Spots 10, 13) that are different from those with the serum
from Non-fish-allergic subject (FIG. 3) and different from the
known salmon allergen proteins, other than 2 (Spots 7, 8) of the 9
spots detected in Example 2, were detected on an immunoblot (FIG.
1B, right panel) of proteins contained in flesh of salmon stained
with the serum from Fish-allergic patient 3. The total 4 spots
detected are indicated in the immunoblot (FIG. 1B, right
panel).
[0569] The molecular weights and isoelectric points of the 2 new
spots detected are as follows. [0570] Spot 10: Molecular weight 200
to 230, pI 4.5 to 5.5 [0571] Spot 13: Molecular weight 45 to 55, pI
4.0 to 5.5
Example 8: Mass Spectrometry and Identification of Antigens (3)
[0572] The amino acid sequences of the antigens that form the 2
spots newly detected were identified by mass spectroscopy in the
same way as in Example 3.
[0573] The mass spectrometry of the spots resulted in the detection
of the following amino acid sequences. [0574] Spot 10: Amino acid
sequences set forth in SEQ ID NOs: 71 to 108 [0575] Spot 13: Amino
acid sequences set forth in SEQ ID NOs: 139 to 142
[0576] Furthermore, the spots were identified as the following
proteins by the analysis of the MS data obtained from the mass
spectrometer for the spots at NCBI. [0577] Spot 10: Myosin heavy
chain, fast skeletal muscle-like (NCBI protein accession number
XP_014039990.1, DNA accession number XM_014184515.1) (amino acid
sequence: SEQ ID NO: 70, encoding nucleotide sequence: SEQ ID NO:
69) [0578] Spot 13: ATP synthase subunit beta, mitochondrial (NCBI
protein accession number XP_014007238.1, DNA accession number
XM_014151763.1) (amino acid sequence: SEQ ID NO: 138, encoding
nucleotide sequence: SEQ ID NO: 137)
Example 9: Confirmation of Antigens in Other Fish Species (2)
[0579] Using flesh of the 6 fish species, horse mackerel, sea eel,
salmon, mackerel, sea bream, and cod, the antigen was confirmed in
the same way as in Example 4. Immunoblots obtained with the serum
from Fish-allergic patient 4 was compared with that obtained with
the control serum from Non-fish-allergic subject. Spots that are
different from those with the serum from Non-fish-allergic subject
(FIG. 3) and correspond to a part of the 14 spots obtained in
salmon with the serum from Fish-allergic patients 1 to 3 were
detected on immunoblots (FIG. 4) of proteins contained in flesh of
each of the 6 fish species stained with the serum from
Fish-allergic patient 4.
[0580] Spots detected in each fish species are as follows. [0581]
Horse mackerel: Spots 1, 3, 4, 8, 10, 11, and 14 [0582] Sea eel:
Spots 1, 4, 10, and 11 [0583] Salmon: Spots 2, 8, and 10-14 [0584]
Mackerel: Spots 4, 8, 10, 11, 13, and 14 [0585] Sea bream: Spots 1,
4, 6, 8, 10, and 13 [0586] Cod: Spots 1, 5, 8, 10, 11, and 13
INDUSTRIAL APPLICABILITY
[0587] The present invention can provide novel antigens of an
allergy to fish, methods and kits for diagnosing an allergy to
fish, a pharmaceutical composition comprising the antigen, and fish
or fish eggs in which the antigen is eliminated or reduced, a
processed product of the fish or fish eggs, or fish which lay the
fish eggs or have hatched from the eggs. The present invention can
further provide a tester for determining the presence or absence of
a fish antigen in an object of interest.
Sequence CWU 1
1
14912685DNAArtificial SequencePREDICTED alpha-actinin-3 1atgacggcaa
tcgaaactca ggtgcaatat ggctcctaca tgacttcgga gcaagtctac 60atgacccaag
aggatgattg ggacagggac cttctgttgg accctgcctg ggagaaacag
120cagcgcaaga ccttcaccgc ctggtgcaac tctcacctgc gtaaagcggg
cacacagatt 180gagaacattg aggaggactt caggaatggg ctcaagctca
tgttgctgtt agaggtcatc 240tcaggtgaaa ggcttcccaa accagacaaa
ggcaaaatgc gtttccacaa aatcgccaac 300gtgaacaagg ccctggattt
catctgcagc aagggagtca agctggtgtc catcggtgcc 360gaggagattg
tggatggtaa tgtgaagatg accctgggga tgatctggac catcattctg
420cgtttcgcca tccaggacat ctctgtagag gagacctctg ctaaggaggg
tctgttgctg 480tggtgccaga ggaagactgc cccctacagg aatgtcaatg
tgcagaactt ccacatcagt 540tggaaggatg gcctggcact gtgtgccctc
atccacagac acagacctga cctcattgac 600tactccaaac tgcgcaagga
tgaccccatg ggcaatctca acactgcctt tgaggtggca 660gagaagtacc
tggacatccc caagatgttg gatgcagaag atattgtgaa caccccgaag
720cccgacgaga aagccatcat gacctatgtg tcctgcttct atcatgcctt
cgctggagcc 780gagcaggccg agacagctgc caataggatc tgcaaggtgt
tggctgtcaa ccaggagaac 840gagaagctca tggaggagta tgagaagctg
gccagtgagc tgctggagtg gatccgtcgc 900accatcccct ggctggagaa
ccgcgtggct gagcagacca tgcgcgccat gcagcagaag 960ctggaggact
tccgtgacta ccgtcgcgtc cacaagcctc ccaaggtgca ggagaagtgt
1020cagctggaga ttaacttcaa caccctgcag accaagctga ggctgagcaa
caggcccgcc 1080ttcatgccct ccgagggcaa gatggtgtcg gacattgcca
atgcctggaa gggtctggag 1140caggtagaga agggctatga ggagtggctg
ctcactgaga tcagacgcct ggagaggctg 1200gaccacctgg ctgagaagtt
caagcagaag tcttccctgc atgagtcctg gacctcaggt 1260aaggtggagc
tgctgtccat gaaggactat gagtctgcct cactgatgga gatccgtgcc
1320ctgatgagga agcacgaggc gtttgagagc gacctggctg cccaccagga
cagagtggag 1380cagattgctg ccatcgccca ggagctcaat gagctggact
atcatgatgc cgtcaccatc 1440aacgcccgct gccagggtat ttgtgaccag
tgggacaacc tgggcacctt gacccagaag 1500aggagagact cactggagcg
tgtggagaag ctgtgggaga ccatcgacca gctgtacctg 1560gagtttgcca
agagagcagc ccccttcaac aactggatgg acggagccat ggaggactta
1620caggacatgt tcattgtgca cagcattgag gagatccaga gtctgatcac
agcccatgac 1680cagttcaagg ccaccctgcc agaggcagat aaggagcgcg
ttgcaaccat gggaatccag 1740aatgagatcg tgaagatcgc tcagacctac
ggcatcaagc tgtcaggagt caacccctac 1800accaaccttt ccccccagga
catcaccgac aaatgggatg ctgtgaaaca cctggtgccc 1860ctcagggatc
agatgctgca ggaggaggtg gccaggcagc agtccaacga gaggctgagg
1920cgccagttcg ctgcccaggc caacatcatc ggaccctgga tccagaccaa
gatggaggag 1980atcagccatg tgtctgtgga catcgccggc tccctggagg
aacagatgag caacctgaag 2040cagtacgagc agaacatcat caactacaag
tgcaacatcg acaagctgga gggagaccac 2100caactcagcc aggagtccct
catctttgac aacaagcaca ccaactacac catggagcat 2160gtgcgtgtgg
gctgggagca gctcctcacc accatcgccc gcaccatcaa cgaggtggag
2220aaccagatcc tgacccgaga tgccaagggc atcagccagg agcagctcaa
cgagttcaga 2280gcctccttca accactttga caggaagaga aatggtatga
tggacccaga tgacttccgt 2340gcctgtctca tctccatggg ctacgatctg
ggtgaggtgg agtttgcccg catcatgacg 2400ctggtggatt ccaacaacac
aggtgtggtg accttccagg ccttcatcga cttcatgacc 2460cgcgagacgg
ccgagacaga caccgccgat caggtcatgg cctcattcaa gatcctggcc
2520tctgacaaga catacatcac agtggaagaa ctgcgcaggg agctgccccc
agagcaggcc 2580gactactgca tcagccgcat gaccagttac atcggcagcg
gcgcaccccc aggcgccctg 2640gactacatct ccttctccag tgccctgtac
ggagagagcg actta 26852895PRTArtificial SequencePREDICTED
alpha-actinin-3 2Met Thr Ala Ile Glu Thr Gln Val Gln Tyr Gly Ser
Tyr Met Thr Ser1 5 10 15Glu Gln Val Tyr Met Thr Gln Glu Asp Asp Trp
Asp Arg Asp Leu Leu 20 25 30Leu Asp Pro Ala Trp Glu Lys Gln Gln Arg
Lys Thr Phe Thr Ala Trp 35 40 45Cys Asn Ser His Leu Arg Lys Ala Gly
Thr Gln Ile Glu Asn Ile Glu 50 55 60Glu Asp Phe Arg Asn Gly Leu Lys
Leu Met Leu Leu Leu Glu Val Ile65 70 75 80Ser Gly Glu Arg Leu Pro
Lys Pro Asp Lys Gly Lys Met Arg Phe His 85 90 95Lys Ile Ala Asn Val
Asn Lys Ala Leu Asp Phe Ile Cys Ser Lys Gly 100 105 110Val Lys Leu
Val Ser Ile Gly Ala Glu Glu Ile Val Asp Gly Asn Val 115 120 125Lys
Met Thr Leu Gly Met Ile Trp Thr Ile Ile Leu Arg Phe Ala Ile 130 135
140Gln Asp Ile Ser Val Glu Glu Thr Ser Ala Lys Glu Gly Leu Leu
Leu145 150 155 160Trp Cys Gln Arg Lys Thr Ala Pro Tyr Arg Asn Val
Asn Val Gln Asn 165 170 175Phe His Ile Ser Trp Lys Asp Gly Leu Ala
Leu Cys Ala Leu Ile His 180 185 190Arg His Arg Pro Asp Leu Ile Asp
Tyr Ser Lys Leu Arg Lys Asp Asp 195 200 205Pro Met Gly Asn Leu Asn
Thr Ala Phe Glu Val Ala Glu Lys Tyr Leu 210 215 220Asp Ile Pro Lys
Met Leu Asp Ala Glu Asp Ile Val Asn Thr Pro Lys225 230 235 240Pro
Asp Glu Lys Ala Ile Met Thr Tyr Val Ser Cys Phe Tyr His Ala 245 250
255Phe Ala Gly Ala Glu Gln Ala Glu Thr Ala Ala Asn Arg Ile Cys Lys
260 265 270Val Leu Ala Val Asn Gln Glu Asn Glu Lys Leu Met Glu Glu
Tyr Glu 275 280 285Lys Leu Ala Ser Glu Leu Leu Glu Trp Ile Arg Arg
Thr Ile Pro Trp 290 295 300Leu Glu Asn Arg Val Ala Glu Gln Thr Met
Arg Ala Met Gln Gln Lys305 310 315 320Leu Glu Asp Phe Arg Asp Tyr
Arg Arg Val His Lys Pro Pro Lys Val 325 330 335Gln Glu Lys Cys Gln
Leu Glu Ile Asn Phe Asn Thr Leu Gln Thr Lys 340 345 350Leu Arg Leu
Ser Asn Arg Pro Ala Phe Met Pro Ser Glu Gly Lys Met 355 360 365Val
Ser Asp Ile Ala Asn Ala Trp Lys Gly Leu Glu Gln Val Glu Lys 370 375
380Gly Tyr Glu Glu Trp Leu Leu Thr Glu Ile Arg Arg Leu Glu Arg
Leu385 390 395 400Asp His Leu Ala Glu Lys Phe Lys Gln Lys Ser Ser
Leu His Glu Ser 405 410 415Trp Thr Ser Gly Lys Val Glu Leu Leu Ser
Met Lys Asp Tyr Glu Ser 420 425 430Ala Ser Leu Met Glu Ile Arg Ala
Leu Met Arg Lys His Glu Ala Phe 435 440 445Glu Ser Asp Leu Ala Ala
His Gln Asp Arg Val Glu Gln Ile Ala Ala 450 455 460Ile Ala Gln Glu
Leu Asn Glu Leu Asp Tyr His Asp Ala Val Thr Ile465 470 475 480Asn
Ala Arg Cys Gln Gly Ile Cys Asp Gln Trp Asp Asn Leu Gly Thr 485 490
495Leu Thr Gln Lys Arg Arg Asp Ser Leu Glu Arg Val Glu Lys Leu Trp
500 505 510Glu Thr Ile Asp Gln Leu Tyr Leu Glu Phe Ala Lys Arg Ala
Ala Pro 515 520 525Phe Asn Asn Trp Met Asp Gly Ala Met Glu Asp Leu
Gln Asp Met Phe 530 535 540Ile Val His Ser Ile Glu Glu Ile Gln Ser
Leu Ile Thr Ala His Asp545 550 555 560Gln Phe Lys Ala Thr Leu Pro
Glu Ala Asp Lys Glu Arg Val Ala Thr 565 570 575Met Gly Ile Gln Asn
Glu Ile Val Lys Ile Ala Gln Thr Tyr Gly Ile 580 585 590Lys Leu Ser
Gly Val Asn Pro Tyr Thr Asn Leu Ser Pro Gln Asp Ile 595 600 605Thr
Asp Lys Trp Asp Ala Val Lys His Leu Val Pro Leu Arg Asp Gln 610 615
620Met Leu Gln Glu Glu Val Ala Arg Gln Gln Ser Asn Glu Arg Leu
Arg625 630 635 640Arg Gln Phe Ala Ala Gln Ala Asn Ile Ile Gly Pro
Trp Ile Gln Thr 645 650 655Lys Met Glu Glu Ile Ser His Val Ser Val
Asp Ile Ala Gly Ser Leu 660 665 670Glu Glu Gln Met Ser Asn Leu Lys
Gln Tyr Glu Gln Asn Ile Ile Asn 675 680 685Tyr Lys Cys Asn Ile Asp
Lys Leu Glu Gly Asp His Gln Leu Ser Gln 690 695 700Glu Ser Leu Ile
Phe Asp Asn Lys His Thr Asn Tyr Thr Met Glu His705 710 715 720Val
Arg Val Gly Trp Glu Gln Leu Leu Thr Thr Ile Ala Arg Thr Ile 725 730
735Asn Glu Val Glu Asn Gln Ile Leu Thr Arg Asp Ala Lys Gly Ile Ser
740 745 750Gln Glu Gln Leu Asn Glu Phe Arg Ala Ser Phe Asn His Phe
Asp Arg 755 760 765Lys Arg Asn Gly Met Met Asp Pro Asp Asp Phe Arg
Ala Cys Leu Ile 770 775 780Ser Met Gly Tyr Asp Leu Gly Glu Val Glu
Phe Ala Arg Ile Met Thr785 790 795 800Leu Val Asp Ser Asn Asn Thr
Gly Val Val Thr Phe Gln Ala Phe Ile 805 810 815Asp Phe Met Thr Arg
Glu Thr Ala Glu Thr Asp Thr Ala Asp Gln Val 820 825 830Met Ala Ser
Phe Lys Ile Leu Ala Ser Asp Lys Thr Tyr Ile Thr Val 835 840 845Glu
Glu Leu Arg Arg Glu Leu Pro Pro Glu Gln Ala Asp Tyr Cys Ile 850 855
860Ser Arg Met Thr Ser Tyr Ile Gly Ser Gly Ala Pro Pro Gly Ala
Leu865 870 875 880Asp Tyr Ile Ser Phe Ser Ser Ala Leu Tyr Gly Glu
Ser Asp Leu 885 890 895314PRTArtificial SequencePREDICTED
alpha-actinin-3 fragment 3Glu Arg Val Ala Thr Met Gly Ile Gln Asn
Glu Ile Val Lys1 5 1043591DNAArtificial SequenceEEF1A2 binding
protein-like 4atgtggaaaa agaaatcaaa gctcacggac cagacggcca
ctggccaagt tgggatcaag 60aagagatcaa aagtccctgg agttatgatc acgcagtatg
tggagaaaat accagatggg 120aaaagccacc ctgacttcac ccgcaagcct
atcgcgttga ccattcagga gggtaaattc 180gccttcttca aagccctggt
tattggagat ccagaaccaa ccgtgacatg gggcagaaat 240aatggagatg
tgtcagatac atcaaaatat gtgacaaaat atgaccctgc tacacgtgag
300cacttatttg agatggccaa tgtaaaacca gaacaagcag acacctacaa
atgctttgca 360gctaatgagt ttggaagagc agtggtcaca gtggtcctca
atgttattga agttgggttc 420aagaaagcac aagcggactc acaggtgcaa
ccagaggcag ctgttgcaga ttttaaatct 480gtattgaaga gaaaaagtaa
aattcagccc aaaatggaaa agaaagaaga tggagaaata 540gatccaagat
tttgggaact cttgataagt gctgacaaga aagactatga gagcctcatg
600ttggagtttg gagtcactga cttccgcttt atgctgaaga cactgaatga
gattaagaag 660gaaagagagg aagagcaagc acagttcatt gaaaacctag
ctaacctgaa acctattgaa 720gttggacccg atggctgtgc aaccttttca
atagacatgg atctcattga acaaagcagc 780aggatcttcc tctacaagga
tggagtgatg attccataca gcaaggagtt gggagataca 840atcaaacaca
gcctaaagat agtgggccga aagtatcagt tcagcataag ggatctgttt
900cctgatgatg ctgggctcta ccaggtggat gttgaggacg taaatgtatt
ctccaccgat 960tttaagattc ccatggtgga cttcctggtc aagattcagg
agtgtaaggc catggagaga 1020gaggatgctg tgtttgagtg tgtcctgtca
cagcccttcg gcaagatcat gtgggttggc 1080aagaacttac cattggaggc
aggggataaa tatgatattg aggtttcaga agacaagctc 1140atccacagac
taatcattaa agacgttgct atggtggaca aaggcatcta tgccgctgtg
1200gcaggaatca aatcttgcaa tgcctttctt gtggttgaag ccgacaaggg
tgaacccggc 1260aaaaagaaac aacgtaaaac cacaagggca ggaggagctg
gagttgacct gacggcgatt 1320gcccaagagc aggcagttaa aaacacggca
gacagagagg tgctgaagga gaaggtgaaa 1380gcaatcaatg acgagagagc
ggctaatgcc actgcagcac ctgagacttc agctgaagct 1440aaagctaaag
ttaaggtggt agaggcttcc caaacaggag ctccaaaaca gggaccggca
1500gttaaagggt ctgaccataa atccgtggat aatgagggat acagcattaa
gagtggactc 1560tcagatgtct ttgctctccg tggcaagaaa ggtgaactgg
tttgtgagat gagccatgaa 1620gttgatgggg cctggttcaa ggatggagag
aagttatcca ccacagatgg aatagccata 1680gtgaaagatg gaacgagaca
cacgatgacc attcatagca gtagtgaaga cgacactgga 1740gtctatcaca
ttgaagcggg gggatttaaa tcagaggcaa aagtcactgt gggagaatta
1800cctgccattg atgctgatga cctccacaag ttttctaagc ctgtgacaat
aaaagtgggc 1860cagaatgcat cctggaagat gccttataca ccacaggaca
acttggaggt gaaatggttt 1920aaggatggca aagagttgaa ggatggtggt
ggggtgaagt tggtgaagga ggtcaagcac 1980agccggctgc tgctccggga
gtgtctgcgt tctgacgctg gggagatcaa gattcaactc 2040aaaaacccct
ttggcactat agaggccaca tctcgactga ttgtccttga caagcccggc
2100ccaccagaag gcccggtgga aatcttggag accacctcca ctgtgattga
gctgcagtgg 2160ggcgctccta aggacgacgg tggctccccg gtgactaact
acatcattga gcgccagcag 2220ctgggacaga ccgtgtggaa gaagatgggt
gatgtcgcag ctgacaaaac cacctacagg 2280gacaggaatg tggtccatgg
gaaactgtac atctacaata tctacgcagt gaacccagag 2340gggaccagtg
atgcactgca gactgaggaa acaatggctg gcgtattgat atttgctggc
2400cgacctggag caccaaaagt ggtcagcgca tcaaagacct gcatcaacct
gaaatgggag 2460cccccagaag atgacggagg aattaagatt gatggctatc
aactggaaaa acgcaaaaag 2520gacacagctc agtggattgc tttgaaccca
gtaactgagc ctattgaagt gctggagtac 2580gcagtgaagg atgttgttga
gggggcggag tatgagttca gggtatcggc catcaacgtt 2640tctggggcgg
gagagttcag tctcccctct gtgatggtga ctgcaaagaa tcccaacatg
2700aggcctacat tcaaagatcc agaggacttc atggtgatca gggcgggaaa
ctctgtgaga 2760atcaaagttc tctatgaggc tgagcctcca ccggagatca
cttggatgaa ggacaatgag 2820cctgtatcca gttttataca gatcatcaac
acagagggct gttcccagct tgtgatcccc 2880tcaacaaaac gctctgattc
aggaaattac accattgtgg ctaagaataa agttggagag 2940gccagctttg
acattgaggt cctagtcaca gatgagccaa agcctcctgg tgcagtggag
3000ctggagcaga ttgtccatgg caaagttatt gtgtcctggg aggcctctcc
agaccaggag 3060ctggacaaca ggctgtatta catggtggcc gagcacgact
ccagcacacg catgtggcac 3120actgtggcag accgcatctt tgacaattca
tacacagcca ataacatcat gcctggaagg 3180gagtaccact tcaaaatcta
tgccaagaac gacatgggca tgtcagaccc ctccatgtca 3240cccacctggg
gcatcaacag caacagaatt cctataaaca caaacacgcc agtggaagtc
3300agctttgaga aaccaccatc tgtcctggtc cctctgaaga tgcactcgcc
accaaaagga 3360ttccagatgt acatgacctg cgccatccgg ggatgcccca
cacccagcgt cacgtggcac 3420ctgaacaacg tctgcatcaa tggtgacagc
aactactaca tcaccaactc gtatggcgtg 3480tgctccatgt acatccttag
ggtcaggccc aaggacgccg gagaatacaa agttgtcgca 3540gttaactcct
tcggcaaggc agaatgctcc actaaacttg ttgttaaaga c
359151197PRTArtificial SequenceEEF1A2 binding protein-like 5Met Trp
Lys Lys Lys Ser Lys Leu Thr Asp Gln Thr Ala Thr Gly Gln1 5 10 15Val
Gly Ile Lys Lys Arg Ser Lys Val Pro Gly Val Met Ile Thr Gln 20 25
30Tyr Val Glu Lys Ile Pro Asp Gly Lys Ser His Pro Asp Phe Thr Arg
35 40 45Lys Pro Ile Ala Leu Thr Ile Gln Glu Gly Lys Phe Ala Phe Phe
Lys 50 55 60Ala Leu Val Ile Gly Asp Pro Glu Pro Thr Val Thr Trp Gly
Arg Asn65 70 75 80Asn Gly Asp Val Ser Asp Thr Ser Lys Tyr Val Thr
Lys Tyr Asp Pro 85 90 95Ala Thr Arg Glu His Leu Phe Glu Met Ala Asn
Val Lys Pro Glu Gln 100 105 110Ala Asp Thr Tyr Lys Cys Phe Ala Ala
Asn Glu Phe Gly Arg Ala Val 115 120 125Val Thr Val Val Leu Asn Val
Ile Glu Val Gly Phe Lys Lys Ala Gln 130 135 140Ala Asp Ser Gln Val
Gln Pro Glu Ala Ala Val Ala Asp Phe Lys Ser145 150 155 160Val Leu
Lys Arg Lys Ser Lys Ile Gln Pro Lys Met Glu Lys Lys Glu 165 170
175Asp Gly Glu Ile Asp Pro Arg Phe Trp Glu Leu Leu Ile Ser Ala Asp
180 185 190Lys Lys Asp Tyr Glu Ser Leu Met Leu Glu Phe Gly Val Thr
Asp Phe 195 200 205Arg Phe Met Leu Lys Thr Leu Asn Glu Ile Lys Lys
Glu Arg Glu Glu 210 215 220Glu Gln Ala Gln Phe Ile Glu Asn Leu Ala
Asn Leu Lys Pro Ile Glu225 230 235 240Val Gly Pro Asp Gly Cys Ala
Thr Phe Ser Ile Asp Met Asp Leu Ile 245 250 255Glu Gln Ser Ser Arg
Ile Phe Leu Tyr Lys Asp Gly Val Met Ile Pro 260 265 270Tyr Ser Lys
Glu Leu Gly Asp Thr Ile Lys His Ser Leu Lys Ile Val 275 280 285Gly
Arg Lys Tyr Gln Phe Ser Ile Arg Asp Leu Phe Pro Asp Asp Ala 290 295
300Gly Leu Tyr Gln Val Asp Val Glu Asp Val Asn Val Phe Ser Thr
Asp305 310 315 320Phe Lys Ile Pro Met Val Asp Phe Leu Val Lys Ile
Gln Glu Cys Lys 325 330 335Ala Met Glu Arg Glu Asp Ala Val Phe Glu
Cys Val Leu Ser Gln Pro 340 345 350Phe Gly Lys Ile Met Trp Val Gly
Lys Asn Leu Pro Leu Glu Ala Gly 355 360 365Asp Lys Tyr Asp Ile Glu
Val Ser Glu Asp Lys Leu Ile His Arg Leu 370 375 380Ile Ile Lys Asp
Val Ala Met Val Asp Lys Gly Ile Tyr Ala Ala Val385 390 395 400Ala
Gly Ile Lys Ser Cys Asn Ala Phe Leu Val Val Glu Ala Asp Lys 405 410
415Gly Glu Pro Gly Lys Lys Lys Gln Arg Lys Thr Thr Arg Ala Gly Gly
420 425 430Ala Gly Val Asp Leu Thr Ala Ile Ala Gln Glu Gln Ala Val
Lys Asn 435 440 445Thr Ala Asp Arg Glu Val Leu Lys Glu Lys Val Lys
Ala Ile Asn Asp 450 455 460Glu Arg Ala Ala Asn Ala Thr Ala Ala Pro
Glu Thr Ser Ala Glu Ala465 470 475 480Lys Ala Lys Val Lys Val Val
Glu Ala Ser Gln Thr
Gly Ala Pro Lys 485 490 495Gln Gly Pro Ala Val Lys Gly Ser Asp His
Lys Ser Val Asp Asn Glu 500 505 510Gly Tyr Ser Ile Lys Ser Gly Leu
Ser Asp Val Phe Ala Leu Arg Gly 515 520 525Lys Lys Gly Glu Leu Val
Cys Glu Met Ser His Glu Val Asp Gly Ala 530 535 540Trp Phe Lys Asp
Gly Glu Lys Leu Ser Thr Thr Asp Gly Ile Ala Ile545 550 555 560Val
Lys Asp Gly Thr Arg His Thr Met Thr Ile His Ser Ser Ser Glu 565 570
575Asp Asp Thr Gly Val Tyr His Ile Glu Ala Gly Gly Phe Lys Ser Glu
580 585 590Ala Lys Val Thr Val Gly Glu Leu Pro Ala Ile Asp Ala Asp
Asp Leu 595 600 605His Lys Phe Ser Lys Pro Val Thr Ile Lys Val Gly
Gln Asn Ala Ser 610 615 620Trp Lys Met Pro Tyr Thr Pro Gln Asp Asn
Leu Glu Val Lys Trp Phe625 630 635 640Lys Asp Gly Lys Glu Leu Lys
Asp Gly Gly Gly Val Lys Leu Val Lys 645 650 655Glu Val Lys His Ser
Arg Leu Leu Leu Arg Glu Cys Leu Arg Ser Asp 660 665 670Ala Gly Glu
Ile Lys Ile Gln Leu Lys Asn Pro Phe Gly Thr Ile Glu 675 680 685Ala
Thr Ser Arg Leu Ile Val Leu Asp Lys Pro Gly Pro Pro Glu Gly 690 695
700Pro Val Glu Ile Leu Glu Thr Thr Ser Thr Val Ile Glu Leu Gln
Trp705 710 715 720Gly Ala Pro Lys Asp Asp Gly Gly Ser Pro Val Thr
Asn Tyr Ile Ile 725 730 735Glu Arg Gln Gln Leu Gly Gln Thr Val Trp
Lys Lys Met Gly Asp Val 740 745 750Ala Ala Asp Lys Thr Thr Tyr Arg
Asp Arg Asn Val Val His Gly Lys 755 760 765Leu Tyr Ile Tyr Asn Ile
Tyr Ala Val Asn Pro Glu Gly Thr Ser Asp 770 775 780Ala Leu Gln Thr
Glu Glu Thr Met Ala Gly Val Leu Ile Phe Ala Gly785 790 795 800Arg
Pro Gly Ala Pro Lys Val Val Ser Ala Ser Lys Thr Cys Ile Asn 805 810
815Leu Lys Trp Glu Pro Pro Glu Asp Asp Gly Gly Ile Lys Ile Asp Gly
820 825 830Tyr Gln Leu Glu Lys Arg Lys Lys Asp Thr Ala Gln Trp Ile
Ala Leu 835 840 845Asn Pro Val Thr Glu Pro Ile Glu Val Leu Glu Tyr
Ala Val Lys Asp 850 855 860Val Val Glu Gly Ala Glu Tyr Glu Phe Arg
Val Ser Ala Ile Asn Val865 870 875 880Ser Gly Ala Gly Glu Phe Ser
Leu Pro Ser Val Met Val Thr Ala Lys 885 890 895Asn Pro Asn Met Arg
Pro Thr Phe Lys Asp Pro Glu Asp Phe Met Val 900 905 910Ile Arg Ala
Gly Asn Ser Val Arg Ile Lys Val Leu Tyr Glu Ala Glu 915 920 925Pro
Pro Pro Glu Ile Thr Trp Met Lys Asp Asn Glu Pro Val Ser Ser 930 935
940Phe Ile Gln Ile Ile Asn Thr Glu Gly Cys Ser Gln Leu Val Ile
Pro945 950 955 960Ser Thr Lys Arg Ser Asp Ser Gly Asn Tyr Thr Ile
Val Ala Lys Asn 965 970 975Lys Val Gly Glu Ala Ser Phe Asp Ile Glu
Val Leu Val Thr Asp Glu 980 985 990Pro Lys Pro Pro Gly Ala Val Glu
Leu Glu Gln Ile Val His Gly Lys 995 1000 1005Val Ile Val Ser Trp
Glu Ala Ser Pro Asp Gln Glu Leu Asp Asn 1010 1015 1020Arg Leu Tyr
Tyr Met Val Ala Glu His Asp Ser Ser Thr Arg Met 1025 1030 1035Trp
His Thr Val Ala Asp Arg Ile Phe Asp Asn Ser Tyr Thr Ala 1040 1045
1050Asn Asn Ile Met Pro Gly Arg Glu Tyr His Phe Lys Ile Tyr Ala
1055 1060 1065Lys Asn Asp Met Gly Met Ser Asp Pro Ser Met Ser Pro
Thr Trp 1070 1075 1080Gly Ile Asn Ser Asn Arg Ile Pro Ile Asn Thr
Asn Thr Pro Val 1085 1090 1095Glu Val Ser Phe Glu Lys Pro Pro Ser
Val Leu Val Pro Leu Lys 1100 1105 1110Met His Ser Pro Pro Lys Gly
Phe Gln Met Tyr Met Thr Cys Ala 1115 1120 1125Ile Arg Gly Cys Pro
Thr Pro Ser Val Thr Trp His Leu Asn Asn 1130 1135 1140Val Cys Ile
Asn Gly Asp Ser Asn Tyr Tyr Ile Thr Asn Ser Tyr 1145 1150 1155Gly
Val Cys Ser Met Tyr Ile Leu Arg Val Arg Pro Lys Asp Ala 1160 1165
1170Gly Glu Tyr Lys Val Val Ala Val Asn Ser Phe Gly Lys Ala Glu
1175 1180 1185Cys Ser Thr Lys Leu Val Val Lys Asp 1190
1195617PRTArtificial SequenceEEF1A2 binding protein-like fragment
6Ala Gln Ala Asp Ser Gln Val Gln Pro Glu Ala Ala Val Ala Asp Phe1 5
10 15Lys710PRTArtificial SequenceEEF1A2 binding protein-like
fragment 7Gly Ile Tyr Ala Ala Val Ala Gly Ile Lys1 5
10821PRTArtificial SequenceEEF1A2 binding protein-like fragment
8Thr Thr Arg Ala Gly Gly Ala Gly Val Asp Leu Thr Ala Ile Ala Gln1 5
10 15Glu Gln Ala Val Lys 2092532DNAArtificial SequenceAlpha-1,4
glucan phosphorylase 9atgtcaaaac cgttgtcaga tcacgataga aagaagcaga
tctccgtgag aggtcttgct 60ggtgtggaaa atgtggcaga actgaaggtc gctttcaaca
ggcatctcca ttttacgctg 120gtcaaggaca gaaatgtggc atccaaacgg
gattactact ttgctctcgc caacaccgtg 180cgtgaccact tggtgggcag
gtggatcaga acccagcaat actactatga gaaagatccc 240aaacgtgtgt
actacatctc cctggagttc tacatgggtc gcaccctgca gaacaccatg
300gtgaacctgg cgctggaaaa cgcctgtgat gaggccatat accagctggg
tctggacatg 360gaggagctgg aggacatgga ggaggacgca ggcctgggaa
acggtggtct tggacgtctt 420gccgcctgct tcctggactc tatggcttct
ctgggtctgg ctgcgtatgg ctacggtatc 480cgctatgagt ttggcatctt
taaccagaag atcgtcaatg gatggcaggt tgaggaggct 540gatgactggc
tgcgttacgg caacccctgg gagaaggccc gacccgagta catgcgcccc
600gtcaagttct atggcagaac cgagcacacc ccagagggtg tgaaatgggt
tgacactcag 660gtagtgttgg ctctgccata tgacacccct atccccgggt
acagaaacaa cattgtcaac 720accatgagac tgtggtctgc aaaggcccca
tgcgacttca acctgaaaga cttcaacgtt 780ggtgggtaca ttcaggctgt
gttggacaga aacctatgcg agaacatttc ccgtgtgctg 840taccccaatg
ataacttctt tgagggcaag gagctgcgtc tgaagcagga gtactttgtg
900gtggccgcca cccttcagga catcgtccgt cgtttcaagg cctctaagtt
tggctccaga 960gagatcgtcc gcacagactt cgcccagctg cccaacaaag
ttgccatcca gctgaatgac 1020actcaccctg ccatggccat tcctgagctg
atgagggtac tggttgatga ggagaagctg 1080gagtgggaca aggcctggga
cgtgtgtgtc cgtacctgtg cctacacaaa ccacaccgtg 1140ctgcctgagg
ccctggagcg ctggcccatt gacctgttcc atcacctgct gccccgtcac
1200ctggagatta tctacgagat caaccgtcgc ttcctgcagt acgtcgcctc
gaagttccct 1260ggcgacaacg accgtctgcg ccgcatgtcc ctgattgagg
agggagaatg caagaaagtc 1320aacatggctc atatgtgtat cgttggatcc
catgctgtca acggcgtggc ccgcatccac 1380tcggagatcc tcgtcgccac
tctgttcaag gacttttatg agttggaccc acacaagttc 1440cagaacaaga
ccaatggcat caccccccgt cgctggctgg ttatgtgcaa ccccggcttg
1500gctgaggtca tcgcagagag aattggagag gagtttgtcc gtgaccttga
ccagctgaag 1560aaactgttga agttcattga tgatgatgct ttcatccgtg
acatagccaa agtcaagcag 1620gagaacaagc tgaagttcgc tgtgcacctg
gaagaacact acaaagtaaa gatcaacccc 1680cagtccatgt ttgacttcca
agtcaaaaga atccacgagt acaagagaca gctgctcaac 1740tgtctgcaca
tgatcaccta ctacaaccgt atcaagaagg agccaaacaa gcactggacc
1800ccaagaacca tcatggtcgg aggaaaggct gccccaggct accacacagc
caagatgatc 1860attcgtctca tcaccgctat cggtgaggtt gtcaaccacg
accccgtgat cggcgaccgc 1920ctcaaagtca tcttcttgga gaactacaga
gtcaccctgg ctgagaaagc catcccctct 1980gctgacctgt ctgagcagat
ctctacagct ggcactgagg cctctggcac tggcaacatg 2040aagttcatgc
tgaatggcgc tctgaccatc ggcaccatgg acggagccaa cgtggagatg
2100gccgaggagg ccggagagaa gaacctcttc atcttcggca tgagagtgga
ggaagtggac 2160gcaatggacg ccggcaaagg ataccacgcc tctgagtact
acaaccgtat tcccgagctg 2220aaacaggcca tggaccagat ctctggtggc
ttcttcagcc ataagcagcc agacctcttc 2280aaggaacttg tggacctgct
gatgcaccac gacaggttca aggtgtttgc tgactacgaa 2340gcctacatca
aaagtcagga taaggtcaac gaactgtaca agaaacccaa ggaatggacc
2400aagatggtga tccataacat tgcaggctgt ggtaaattct ccagcgaccg
caccatttcc 2460cagtacgccc gggagatctg gggcatggag cccagcctgg
agaagatccc tgcccccgat 2520gagcaactca aa 253210844PRTArtificial
SequenceAlpha-1,4 glucan phosphorylase 10Met Ser Lys Pro Leu Ser
Asp His Asp Arg Lys Lys Gln Ile Ser Val1 5 10 15Arg Gly Leu Ala Gly
Val Glu Asn Val Ala Glu Leu Lys Val Ala Phe 20 25 30Asn Arg His Leu
His Phe Thr Leu Val Lys Asp Arg Asn Val Ala Ser 35 40 45Lys Arg Asp
Tyr Tyr Phe Ala Leu Ala Asn Thr Val Arg Asp His Leu 50 55 60Val Gly
Arg Trp Ile Arg Thr Gln Gln Tyr Tyr Tyr Glu Lys Asp Pro65 70 75
80Lys Arg Val Tyr Tyr Ile Ser Leu Glu Phe Tyr Met Gly Arg Thr Leu
85 90 95Gln Asn Thr Met Val Asn Leu Ala Leu Glu Asn Ala Cys Asp Glu
Ala 100 105 110Ile Tyr Gln Leu Gly Leu Asp Met Glu Glu Leu Glu Asp
Met Glu Glu 115 120 125Asp Ala Gly Leu Gly Asn Gly Gly Leu Gly Arg
Leu Ala Ala Cys Phe 130 135 140Leu Asp Ser Met Ala Ser Leu Gly Leu
Ala Ala Tyr Gly Tyr Gly Ile145 150 155 160Arg Tyr Glu Phe Gly Ile
Phe Asn Gln Lys Ile Val Asn Gly Trp Gln 165 170 175Val Glu Glu Ala
Asp Asp Trp Leu Arg Tyr Gly Asn Pro Trp Glu Lys 180 185 190Ala Arg
Pro Glu Tyr Met Arg Pro Val Lys Phe Tyr Gly Arg Thr Glu 195 200
205His Thr Pro Glu Gly Val Lys Trp Val Asp Thr Gln Val Val Leu Ala
210 215 220Leu Pro Tyr Asp Thr Pro Ile Pro Gly Tyr Arg Asn Asn Ile
Val Asn225 230 235 240Thr Met Arg Leu Trp Ser Ala Lys Ala Pro Cys
Asp Phe Asn Leu Lys 245 250 255Asp Phe Asn Val Gly Gly Tyr Ile Gln
Ala Val Leu Asp Arg Asn Leu 260 265 270Cys Glu Asn Ile Ser Arg Val
Leu Tyr Pro Asn Asp Asn Phe Phe Glu 275 280 285Gly Lys Glu Leu Arg
Leu Lys Gln Glu Tyr Phe Val Val Ala Ala Thr 290 295 300Leu Gln Asp
Ile Val Arg Arg Phe Lys Ala Ser Lys Phe Gly Ser Arg305 310 315
320Glu Ile Val Arg Thr Asp Phe Ala Gln Leu Pro Asn Lys Val Ala Ile
325 330 335Gln Leu Asn Asp Thr His Pro Ala Met Ala Ile Pro Glu Leu
Met Arg 340 345 350Val Leu Val Asp Glu Glu Lys Leu Glu Trp Asp Lys
Ala Trp Asp Val 355 360 365Cys Val Arg Thr Cys Ala Tyr Thr Asn His
Thr Val Leu Pro Glu Ala 370 375 380Leu Glu Arg Trp Pro Ile Asp Leu
Phe His His Leu Leu Pro Arg His385 390 395 400Leu Glu Ile Ile Tyr
Glu Ile Asn Arg Arg Phe Leu Gln Tyr Val Ala 405 410 415Ser Lys Phe
Pro Gly Asp Asn Asp Arg Leu Arg Arg Met Ser Leu Ile 420 425 430Glu
Glu Gly Glu Cys Lys Lys Val Asn Met Ala His Met Cys Ile Val 435 440
445Gly Ser His Ala Val Asn Gly Val Ala Arg Ile His Ser Glu Ile Leu
450 455 460Val Ala Thr Leu Phe Lys Asp Phe Tyr Glu Leu Asp Pro His
Lys Phe465 470 475 480Gln Asn Lys Thr Asn Gly Ile Thr Pro Arg Arg
Trp Leu Val Met Cys 485 490 495Asn Pro Gly Leu Ala Glu Val Ile Ala
Glu Arg Ile Gly Glu Glu Phe 500 505 510Val Arg Asp Leu Asp Gln Leu
Lys Lys Leu Leu Lys Phe Ile Asp Asp 515 520 525Asp Ala Phe Ile Arg
Asp Ile Ala Lys Val Lys Gln Glu Asn Lys Leu 530 535 540Lys Phe Ala
Val His Leu Glu Glu His Tyr Lys Val Lys Ile Asn Pro545 550 555
560Gln Ser Met Phe Asp Phe Gln Val Lys Arg Ile His Glu Tyr Lys Arg
565 570 575Gln Leu Leu Asn Cys Leu His Met Ile Thr Tyr Tyr Asn Arg
Ile Lys 580 585 590Lys Glu Pro Asn Lys His Trp Thr Pro Arg Thr Ile
Met Val Gly Gly 595 600 605Lys Ala Ala Pro Gly Tyr His Thr Ala Lys
Met Ile Ile Arg Leu Ile 610 615 620Thr Ala Ile Gly Glu Val Val Asn
His Asp Pro Val Ile Gly Asp Arg625 630 635 640Leu Lys Val Ile Phe
Leu Glu Asn Tyr Arg Val Thr Leu Ala Glu Lys 645 650 655Ala Ile Pro
Ser Ala Asp Leu Ser Glu Gln Ile Ser Thr Ala Gly Thr 660 665 670Glu
Ala Ser Gly Thr Gly Asn Met Lys Phe Met Leu Asn Gly Ala Leu 675 680
685Thr Ile Gly Thr Met Asp Gly Ala Asn Val Glu Met Ala Glu Glu Ala
690 695 700Gly Glu Lys Asn Leu Phe Ile Phe Gly Met Arg Val Glu Glu
Val Asp705 710 715 720Ala Met Asp Ala Gly Lys Gly Tyr His Ala Ser
Glu Tyr Tyr Asn Arg 725 730 735Ile Pro Glu Leu Lys Gln Ala Met Asp
Gln Ile Ser Gly Gly Phe Phe 740 745 750Ser His Lys Gln Pro Asp Leu
Phe Lys Glu Leu Val Asp Leu Leu Met 755 760 765His His Asp Arg Phe
Lys Val Phe Ala Asp Tyr Glu Ala Tyr Ile Lys 770 775 780Ser Gln Asp
Lys Val Asn Glu Leu Tyr Lys Lys Pro Lys Glu Trp Thr785 790 795
800Lys Met Val Ile His Asn Ile Ala Gly Cys Gly Lys Phe Ser Ser Asp
805 810 815Arg Thr Ile Ser Gln Tyr Ala Arg Glu Ile Trp Gly Met Glu
Pro Ser 820 825 830Leu Glu Lys Ile Pro Ala Pro Asp Glu Gln Leu Lys
835 840119PRTArtificial SequenceAlpha-1,4 glucan phosphorylase
fragment 11Ile Pro Ala Pro Asp Glu Gln Leu Lys1 51217PRTArtificial
SequenceAlpha-1,4 glucan phosphorylase fragment 12Gln Ile Ser Val
Arg Gly Leu Ala Gly Val Glu Asn Val Ala Glu Leu1 5 10
15Lys1325PRTArtificial SequenceAlpha-1,4 glucan phosphorylase
fragment 13Ala Ile Pro Ser Ala Asp Leu Ser Glu Gln Ile Ser Thr Ala
Gly Thr1 5 10 15Glu Ala Ser Gly Thr Gly Asn Met Lys 20
25149PRTArtificial SequenceAlpha-1,4 glucan phosphorylase fragment
14Asp Phe Tyr Glu Leu Asp Pro His Lys1 51510PRTArtificial
SequenceAlpha-1,4 glucan phosphorylase fragment 15Phe Ala Val His
Leu Glu Glu His Tyr Lys1 5 101615PRTArtificial SequenceAlpha-1,4
glucan phosphorylase fragment 16Gly Tyr His Ala Ser Glu Tyr Tyr Asn
Arg Ile Pro Glu Leu Lys1 5 10 15179PRTArtificial SequenceAlpha-1,4
glucan phosphorylase fragment 17Ile Pro Ala Pro Asp Glu Gln Leu
Lys1 5182574DNAArtificial SequenceElongation factor 2 18atggtgaact
ttacagtgga ccagatccgt gccatcatgg acaagaaatc caacattcgt 60aacatgtctg
tgatcgctca cgtggaccac ggcaagtcca cgctgaccga ctccctggtg
120tctaaagccg ggatcatcgc ggggtctcgc gccggagaga cacgcttcac
agacactcgc 180aaagacgagc aggagcgctg tattaccatc aagtccacgg
ctatctcgat gtactatgag 240ctgggggaaa acgacatggc cttcatcaag
cagtctaagg atgggcttgg cttcctcatc 300aacctgattg actcaccggg
ccatgtggac ttctcctctg aggtcacagc cgcccttagg 360gtcaccgacg
gcgccctggt ggtggttgac tgcgtctcag gtgtgtgtgt gcagacagag
420accgtgttga ggcaggccat tgctgagcgc atcaagccag tgctgatgat
gaacaagatg 480gaccgggccc tgctggagct gcagctggag cctgaggacc
tgttccagac cttccagcgc 540atcgtggaga atgtcaacgt catcattgcc
acctacggag aggatgaagc gggaccaatg 600ggtgccatca tgattgaccc
tgtgattggt accgtggggt ttgggtctgg cctccacggc 660tgggccttca
ctctaaagca gtttgctgag atgtacgtga caaagttttc tgccggcaaa
720gacacccagc tgggatcggc ggagaggtgt aagaaggtgg aggacatgat
gaagaagctg 780tggggggaga ggttttttga cccagccact gggaagttca
gtaagtccaa cctcggccct 840gacggtaaga agctgccccg caccttctct
cagctggtcc tggaccctat cttcaaggta 900tttgatgcca tcatgaactt
caagaaggat gagacagcca agctgataga gaagctggac 960atcaagctgg
actctgagga caaggagaag gagggcaagc ccctgttgaa ggcagtgatg
1020cgtcgctggc tcccagccgg agaagccctg ctccagatga tcaccatcca
cctgccctcc 1080cccgtcacgg cccagaagta ccgctgtgag ctgctctacg
agggaccagg agacgacgag 1140gccgccatgg gtatcaagaa ctgcgacccc
aaggctcccc tgatgatgta catatctaag 1200atggtgccca ccacagacaa
gggtcgcttc tatgcctttg gccgtgtgtt ctctggctgt 1260gtgtccaccg
gtctgaaggt gcgcatcatg ggaccaaact tcacccctgg gaagaaggaa
1320gacctctaca tcaagcccat ccagaggacc attctgatga tggggcgtta
tgtggagccc 1380attgaggatg taccatgtgg gaacatcgtt gggctggttg
gagttgacca gtatctgatt 1440aagactggga
ccatcaccac ctttgaacag gcccacaaca tgcgtgtgat gaagttcagc
1500gtcagccctg tggtgagggt ggctgtggag gccaagaacc ctgctgacct
gcccaagctg 1560gtggaggggc tgaagcgtct ggccaagtct gaccccatgg
tgcagtgtat catcgaggag 1620tctggagagc acatcatcgc cggggccgga
gagcttcatc tggagatctg tctcaaggat 1680ctggaggagg accacgccgg
cattcccctg aagaaatctg atccagtggt gtcctacagg 1740gagactgtgt
ctgaggagtc tgaagtgatg tgtctatcca agtcccctaa caagcacaac
1800cgtctgtaca tgcgggctaa acctttccct gacggcctgg ccgaggacat
cgagaagggg 1860gacgtcagcc cccgacagga gctgaaaatc cgcgcccgtt
tcctggctga caagtacgag 1920tgggacgtgt cggaggcccg taagatctgg
tgcttcggcc ctgacggtac cggtcccaac 1980ctgctgatgg atgttaccaa
gggagtccag tacctgaatg agatcaagga cagtgttgtg 2040gctggcttcc
agtgggctgt caaggagggt gtgttgtgtg aggagaacat gcgtgcagtc
2100cgcttcgaca tccacgacgt gaccctgcac acagacgcaa ttcaccgcgg
tggcggacag 2160atcatcccca cggcccgcag agtgctgtat gcctgccagc
tcaccgccca gccacgactc 2220atggagccgg tctacttagt ggagatccag
tgcccagagc aggtagtggg tgggatctac 2280ggcgtgctga acaggaagcg
aggccatgtg ttcgaggagt cccaggtgat gggcacgccc 2340atgttcatcg
tcaaggccta cctgcctgtc aacgagtcat ttgggttcac cgctgacctg
2400cgctccaaca cgggcggcca ggctttcccc cagtgtgtgt ttgatcactg
gcagatcctc 2460cagggagatc cccaggaccc caccaccaag accgccattg
tggtggccga gaccaggaaa 2520cgcaaggggc tgaaagaggg catcccggcc
ctggacaact acctggacaa attg 257419858PRTArtificial
SequenceElongation factor 2 19Met Val Asn Phe Thr Val Asp Gln Ile
Arg Ala Ile Met Asp Lys Lys1 5 10 15Ser Asn Ile Arg Asn Met Ser Val
Ile Ala His Val Asp His Gly Lys 20 25 30Ser Thr Leu Thr Asp Ser Leu
Val Ser Lys Ala Gly Ile Ile Ala Gly 35 40 45Ser Arg Ala Gly Glu Thr
Arg Phe Thr Asp Thr Arg Lys Asp Glu Gln 50 55 60Glu Arg Cys Ile Thr
Ile Lys Ser Thr Ala Ile Ser Met Tyr Tyr Glu65 70 75 80Leu Gly Glu
Asn Asp Met Ala Phe Ile Lys Gln Ser Lys Asp Gly Leu 85 90 95Gly Phe
Leu Ile Asn Leu Ile Asp Ser Pro Gly His Val Asp Phe Ser 100 105
110Ser Glu Val Thr Ala Ala Leu Arg Val Thr Asp Gly Ala Leu Val Val
115 120 125Val Asp Cys Val Ser Gly Val Cys Val Gln Thr Glu Thr Val
Leu Arg 130 135 140Gln Ala Ile Ala Glu Arg Ile Lys Pro Val Leu Met
Met Asn Lys Met145 150 155 160Asp Arg Ala Leu Leu Glu Leu Gln Leu
Glu Pro Glu Asp Leu Phe Gln 165 170 175Thr Phe Gln Arg Ile Val Glu
Asn Val Asn Val Ile Ile Ala Thr Tyr 180 185 190Gly Glu Asp Glu Ala
Gly Pro Met Gly Ala Ile Met Ile Asp Pro Val 195 200 205Ile Gly Thr
Val Gly Phe Gly Ser Gly Leu His Gly Trp Ala Phe Thr 210 215 220Leu
Lys Gln Phe Ala Glu Met Tyr Val Thr Lys Phe Ser Ala Gly Lys225 230
235 240Asp Thr Gln Leu Gly Ser Ala Glu Arg Cys Lys Lys Val Glu Asp
Met 245 250 255Met Lys Lys Leu Trp Gly Glu Arg Phe Phe Asp Pro Ala
Thr Gly Lys 260 265 270Phe Ser Lys Ser Asn Leu Gly Pro Asp Gly Lys
Lys Leu Pro Arg Thr 275 280 285Phe Ser Gln Leu Val Leu Asp Pro Ile
Phe Lys Val Phe Asp Ala Ile 290 295 300Met Asn Phe Lys Lys Asp Glu
Thr Ala Lys Leu Ile Glu Lys Leu Asp305 310 315 320Ile Lys Leu Asp
Ser Glu Asp Lys Glu Lys Glu Gly Lys Pro Leu Leu 325 330 335Lys Ala
Val Met Arg Arg Trp Leu Pro Ala Gly Glu Ala Leu Leu Gln 340 345
350Met Ile Thr Ile His Leu Pro Ser Pro Val Thr Ala Gln Lys Tyr Arg
355 360 365Cys Glu Leu Leu Tyr Glu Gly Pro Gly Asp Asp Glu Ala Ala
Met Gly 370 375 380Ile Lys Asn Cys Asp Pro Lys Ala Pro Leu Met Met
Tyr Ile Ser Lys385 390 395 400Met Val Pro Thr Thr Asp Lys Gly Arg
Phe Tyr Ala Phe Gly Arg Val 405 410 415Phe Ser Gly Cys Val Ser Thr
Gly Leu Lys Val Arg Ile Met Gly Pro 420 425 430Asn Phe Thr Pro Gly
Lys Lys Glu Asp Leu Tyr Ile Lys Pro Ile Gln 435 440 445Arg Thr Ile
Leu Met Met Gly Arg Tyr Val Glu Pro Ile Glu Asp Val 450 455 460Pro
Cys Gly Asn Ile Val Gly Leu Val Gly Val Asp Gln Tyr Leu Ile465 470
475 480Lys Thr Gly Thr Ile Thr Thr Phe Glu Gln Ala His Asn Met Arg
Val 485 490 495Met Lys Phe Ser Val Ser Pro Val Val Arg Val Ala Val
Glu Ala Lys 500 505 510Asn Pro Ala Asp Leu Pro Lys Leu Val Glu Gly
Leu Lys Arg Leu Ala 515 520 525Lys Ser Asp Pro Met Val Gln Cys Ile
Ile Glu Glu Ser Gly Glu His 530 535 540Ile Ile Ala Gly Ala Gly Glu
Leu His Leu Glu Ile Cys Leu Lys Asp545 550 555 560Leu Glu Glu Asp
His Ala Gly Ile Pro Leu Lys Lys Ser Asp Pro Val 565 570 575Val Ser
Tyr Arg Glu Thr Val Ser Glu Glu Ser Glu Val Met Cys Leu 580 585
590Ser Lys Ser Pro Asn Lys His Asn Arg Leu Tyr Met Arg Ala Lys Pro
595 600 605Phe Pro Asp Gly Leu Ala Glu Asp Ile Glu Lys Gly Asp Val
Ser Pro 610 615 620Arg Gln Glu Leu Lys Ile Arg Ala Arg Phe Leu Ala
Asp Lys Tyr Glu625 630 635 640Trp Asp Val Ser Glu Ala Arg Lys Ile
Trp Cys Phe Gly Pro Asp Gly 645 650 655Thr Gly Pro Asn Leu Leu Met
Asp Val Thr Lys Gly Val Gln Tyr Leu 660 665 670Asn Glu Ile Lys Asp
Ser Val Val Ala Gly Phe Gln Trp Ala Val Lys 675 680 685Glu Gly Val
Leu Cys Glu Glu Asn Met Arg Ala Val Arg Phe Asp Ile 690 695 700His
Asp Val Thr Leu His Thr Asp Ala Ile His Arg Gly Gly Gly Gln705 710
715 720Ile Ile Pro Thr Ala Arg Arg Val Leu Tyr Ala Cys Gln Leu Thr
Ala 725 730 735Gln Pro Arg Leu Met Glu Pro Val Tyr Leu Val Glu Ile
Gln Cys Pro 740 745 750Glu Gln Val Val Gly Gly Ile Tyr Gly Val Leu
Asn Arg Lys Arg Gly 755 760 765His Val Phe Glu Glu Ser Gln Val Met
Gly Thr Pro Met Phe Ile Val 770 775 780Lys Ala Tyr Leu Pro Val Asn
Glu Ser Phe Gly Phe Thr Ala Asp Leu785 790 795 800Arg Ser Asn Thr
Gly Gly Gln Ala Phe Pro Gln Cys Val Phe Asp His 805 810 815Trp Gln
Ile Leu Gln Gly Asp Pro Gln Asp Pro Thr Thr Lys Thr Ala 820 825
830Ile Val Val Ala Glu Thr Arg Lys Arg Lys Gly Leu Lys Glu Gly Ile
835 840 845Pro Ala Leu Asp Asn Tyr Leu Asp Lys Leu 850
8552010PRTArtificial SequenceElongation factor 2 fragment 20Thr Ala
Ile Val Val Ala Glu Thr Arg Lys1 5 102112PRTArtificial
SequenceElongation factor 2 fragment 21Asp Leu Glu Glu Asp His Ala
Gly Ile Pro Leu Lys1 5 102213PRTArtificial SequenceElongation
factor 2 fragment 22Asp Leu Glu Glu Asp His Ala Gly Ile Pro Leu Lys
Lys1 5 102312PRTArtificial SequenceElongation factor 2 fragment
23Asp Ser Val Val Ala Gly Phe Gln Trp Ala Val Lys1 5
102410PRTArtificial SequenceElongation factor 2 fragment 24Gly Asp
Val Ser Pro Arg Gln Glu Leu Lys1 5 10251989DNAArtificial
SequenceHeat shock cognate 70 kDa protein 25atgtcaaagg gaccagcagt
tggcattgac ctgggcacca cctactcctg tgtgggtgtg 60tttcagcatg gcaaagtgga
gatcatagcc aacgaccagg gaaacaggac cacacccagt 120tatgtcgcct
tcacagacac agaaaggctg attggggacg cagccaagaa ccaagtggcc
180atgaacccca caaacacagt gtttgatgct aagcggctga tagggcggaa
gtttgacgac 240agtgtcgtcc aggcagacat gaaacactgg ccgtttacag
tgatcaacga ctcgacacgg 300cccaaggtcc aagtggagta caagggagag
accaaggcct tctacccaga ggagatctcc 360tccatggtgc tggtcaagat
gaaggagatt gcagaggcct acctgggcaa gactataacc 420aatgcagtgg
tcactgtgcc agcttacttc aacgactctc agcgccaggc caccaaagat
480gcagggacta tctcaggact caacgtactc cgcatcatca atgagccaac
tgctgctgct 540atcgcctatg gcctggacaa gaaggtggga gtggagagaa
acgtcctaat ctttgaccta 600ggcggaggta cgtttgatgt gtctatcctg
accatcgaag acgggatctt tgaggtgaag 660tccacggccg gagacaccca
tcttggagga gaggacttcg acaaccgcat ggtcaaccac 720ttcatctctg
agttcaagcg caaatacaag aaggacatca gcgataacaa gagggccgtg
780cgtcgtctgc gcaccgcctg tgaacgtgcc aagcgcaccc tgtcctccag
tacccaggcc 840agcattgaga ttgactctct gtatgagggc gtcgacttct
acacctccat caccagggct 900cgctttgagg agctgaacgc tgacctgttc
agaggcaccc tggaccccgt ggagaagtct 960ctgagggacg ccaagatgga
caaggcccag gttcacgaca tcgtcctagt gggaggctcc 1020acacgcatcc
ccaagatcca aaagttgctg caggacttct tcaatgggaa ggagctcaac
1080aagagcatca accctgatga ggcggttgcc tatggtgcag ctgttcaggc
ggccatcttg 1140tccggagaca agtctgagaa cgtgcaggac ctgctgctgc
tggacgtcac gccactgtcc 1200ctgggcattg agacggccgg aggggtcatg
accgtgctca tcaagaggaa caccaccatc 1260cccaccaagc agacgcagac
cttcacaaca tactcagaca accagcctgg ggtgctcatt 1320caggtatatg
agggggaaag agccatgacc aaagacaaca acctactggg gaagtttgag
1380ttgtgtggga ttccaccagc cccccggggt gtgcctcaga tcgaggtcac
ctttgacatt 1440gacgccaacg gcatcatgaa cgtgtctgcc gctgacaaga
gcaccggcaa ggagaataag 1500atcaccatca ccaatgacaa aggtcgtctg
agtaaggagg acatagagcg catggtccag 1560gaggctgaac agtacaaagc
tgcggacgat gtccagaggg acaaggtggc gtccaagaat 1620ggcctggagt
catacgcctt caacatgaag tccaccgtgg aggacgagaa gctcaagggc
1680aagctcagcg acgaggacaa acagaagatc ctggacaaat gcaatgaggt
catcagctgg 1740ctggacaaga accagtctgc agagaaggag gagtttgagc
accaccagaa ggagctggag 1800aaggtctgta accccatcat caccaagctg
taccagggtg ctggggggat gcctggaggg 1860atgcctggag ggatgcctgg
agggatgcct ggagggatgc ctggggggat gcctggaggg 1920atgcccgggg
ctggtggtgc cgcacccgga ggaggtggat cctctggacc aacaattgag
1980gaagtcgac 198926663PRTArtificial SequenceHeat shock cognate 70
kDa protein 26Met Ser Lys Gly Pro Ala Val Gly Ile Asp Leu Gly Thr
Thr Tyr Ser1 5 10 15Cys Val Gly Val Phe Gln His Gly Lys Val Glu Ile
Ile Ala Asn Asp 20 25 30Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala
Phe Thr Asp Thr Glu 35 40 45Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln
Val Ala Met Asn Pro Thr 50 55 60Asn Thr Val Phe Asp Ala Lys Arg Leu
Ile Gly Arg Lys Phe Asp Asp65 70 75 80Ser Val Val Gln Ala Asp Met
Lys His Trp Pro Phe Thr Val Ile Asn 85 90 95Asp Ser Thr Arg Pro Lys
Val Gln Val Glu Tyr Lys Gly Glu Thr Lys 100 105 110Ala Phe Tyr Pro
Glu Glu Ile Ser Ser Met Val Leu Val Lys Met Lys 115 120 125Glu Ile
Ala Glu Ala Tyr Leu Gly Lys Thr Ile Thr Asn Ala Val Val 130 135
140Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys
Asp145 150 155 160Ala Gly Thr Ile Ser Gly Leu Asn Val Leu Arg Ile
Ile Asn Glu Pro 165 170 175Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp
Lys Lys Val Gly Val Glu 180 185 190Arg Asn Val Leu Ile Phe Asp Leu
Gly Gly Gly Thr Phe Asp Val Ser 195 200 205Ile Leu Thr Ile Glu Asp
Gly Ile Phe Glu Val Lys Ser Thr Ala Gly 210 215 220Asp Thr His Leu
Gly Gly Glu Asp Phe Asp Asn Arg Met Val Asn His225 230 235 240Phe
Ile Ser Glu Phe Lys Arg Lys Tyr Lys Lys Asp Ile Ser Asp Asn 245 250
255Lys Arg Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg Ala Lys Arg
260 265 270Thr Leu Ser Ser Ser Thr Gln Ala Ser Ile Glu Ile Asp Ser
Leu Tyr 275 280 285Glu Gly Val Asp Phe Tyr Thr Ser Ile Thr Arg Ala
Arg Phe Glu Glu 290 295 300Leu Asn Ala Asp Leu Phe Arg Gly Thr Leu
Asp Pro Val Glu Lys Ser305 310 315 320Leu Arg Asp Ala Lys Met Asp
Lys Ala Gln Val His Asp Ile Val Leu 325 330 335Val Gly Gly Ser Thr
Arg Ile Pro Lys Ile Gln Lys Leu Leu Gln Asp 340 345 350Phe Phe Asn
Gly Lys Glu Leu Asn Lys Ser Ile Asn Pro Asp Glu Ala 355 360 365Val
Ala Tyr Gly Ala Ala Val Gln Ala Ala Ile Leu Ser Gly Asp Lys 370 375
380Ser Glu Asn Val Gln Asp Leu Leu Leu Leu Asp Val Thr Pro Leu
Ser385 390 395 400Leu Gly Ile Glu Thr Ala Gly Gly Val Met Thr Val
Leu Ile Lys Arg 405 410 415Asn Thr Thr Ile Pro Thr Lys Gln Thr Gln
Thr Phe Thr Thr Tyr Ser 420 425 430Asp Asn Gln Pro Gly Val Leu Ile
Gln Val Tyr Glu Gly Glu Arg Ala 435 440 445Met Thr Lys Asp Asn Asn
Leu Leu Gly Lys Phe Glu Leu Cys Gly Ile 450 455 460Pro Pro Ala Pro
Arg Gly Val Pro Gln Ile Glu Val Thr Phe Asp Ile465 470 475 480Asp
Ala Asn Gly Ile Met Asn Val Ser Ala Ala Asp Lys Ser Thr Gly 485 490
495Lys Glu Asn Lys Ile Thr Ile Thr Asn Asp Lys Gly Arg Leu Ser Lys
500 505 510Glu Asp Ile Glu Arg Met Val Gln Glu Ala Glu Gln Tyr Lys
Ala Ala 515 520 525Asp Asp Val Gln Arg Asp Lys Val Ala Ser Lys Asn
Gly Leu Glu Ser 530 535 540Tyr Ala Phe Asn Met Lys Ser Thr Val Glu
Asp Glu Lys Leu Lys Gly545 550 555 560Lys Leu Ser Asp Glu Asp Lys
Gln Lys Ile Leu Asp Lys Cys Asn Glu 565 570 575Val Ile Ser Trp Leu
Asp Lys Asn Gln Ser Ala Glu Lys Glu Glu Phe 580 585 590Glu His His
Gln Lys Glu Leu Glu Lys Val Cys Asn Pro Ile Ile Thr 595 600 605Lys
Leu Tyr Gln Gly Ala Gly Gly Met Pro Gly Gly Met Pro Gly Gly 610 615
620Met Pro Gly Gly Met Pro Gly Gly Met Pro Gly Gly Met Pro Gly
Gly625 630 635 640Met Pro Gly Ala Gly Gly Ala Ala Pro Gly Gly Gly
Gly Ser Ser Gly 645 650 655Pro Thr Ile Glu Glu Val Asp
6602714PRTArtificial SequenceHeat shock cognate 70 kDa protein
fragment 27Ala Phe Tyr Pro Glu Glu Ile Ser Ser Met Val Leu Val Lys1
5 102814PRTArtificial SequenceHeat shock cognate 70 kDa protein
fragment 28Glu Asp Ile Glu Arg Met Val Gln Glu Ala Glu Gln Tyr Lys1
5 102914PRTArtificial SequenceHeat shock cognate 70 kDa protein
fragment 29His Trp Pro Phe Thr Val Ile Asn Asp Ser Thr Arg Pro Lys1
5 103014PRTArtificial SequenceHeat shock cognate 70 kDa protein
fragment 30Asn Gln Ser Ala Glu Lys Glu Glu Phe Glu His His Gln Lys1
5 103122PRTArtificial SequenceHeat shock cognate 70 kDa protein
fragment 31Thr Ile Thr Asn Ala Val Val Thr Val Pro Ala Tyr Phe Asn
Asp Ser1 5 10 15Gln Arg Gln Ala Thr Lys 20322061DNAArtificial
SequenceSerotransferrin 32atgaaactgc ttctcgtctc agcgctgctg
gggtgcttcg ctacggtgta tgctgcccca 60gctgagggaa tggttagatg gtgcgtaaag
tcggaaaaag agctgaagaa atgccacgat 120ctcgcagcca atgtggcggg
gttttcatgc gtgaggaggg acgactctct tgaatgcatc 180caagccatca
agagggaaga ggctgatgct atcactctgg atggaggaga tatttacata
240gctggcctcc acaactacaa cctgcagccc atcattgcag aggactatgg
tgaggactct 300gacacctgct attatgctgt ggccgtggcc aaaaagggca
ctgaatttgg gttcctcgac 360ctccgtggca agaagtcctg ccacaccggg
ttgggcaaat ctgcaggctg gaacattccc 420atcggtaccc tggtgactgt
gggccagatc caatgggccg gcatcgagga cagacctgtg 480gagtcggcgg
tgagcgactt cttcaatgcc agctgtgctc caggagccaa tagggattct
540caactgtgcc agttgtgtat gggagactgc tccagatctc acaacgagcc
ctactatgac 600tattccgggg ccttccagtg cctgaaggat ggagcaggag
aggttgcctt catcaagcac 660ctgactgtac ctgccgcaga gaaggcaagc
tatgagttgc tgtgcaagga taacaccaga 720gctcccatcg acagctacaa
gacctgccac ctggccagag tacccgccca cgctgtggtc 780agccgcaagg
accccaggct ggccaatctc atctacagca agctgatggc cgtcacgaac
840ttcaacctgt tctcctccga tggttatgct gccaagaacc tgatgttcaa
ggactccact 900cagaatctag tacagctgcc aatgaccacc gactccttcc
tctacctggg agctgagtac 960atgagcacta tacgctccct gacaaaagcg
caggccacag gtgtcacctc cagggccatc 1020aaatggtgtg ccgtgggcca
taaggagaag gtcaagtgtg
acgcctggac aatcaacagc 1080ttcacagatg gtgactccag gatcgaatgc
caggacgcac ccacagtgga tgaatgcatc 1140aagaagatca tgcgtaaaga
ggcagatgcc atagcggtgg atggtgggga ggtgttcact 1200gctggaaaat
gtggtctggt ccctgtcatg gtggagcagt atgatgaagt tcggtgcagc
1260gcccctggtg aggcgtcatc ctactttgcg gtggcggtgg caaagagggg
atctgggttg 1320acctggacaa ccctgaaggg caagaggtca tgccacaccg
gcttgggcag gaccgcaggc 1380tggaacatac ccatgggtct tatccatagg
aggactatga actgcgactt caccacatac 1440ttcagtaagg gctgtgctcc
tggatttgag gtggactctc ccttctgtgc ccagtgtaag 1500ggcagtggga
agtccgtggg aggagatggg tccaagtgca aagccagctc tgaagagcag
1560tactacggct ataacggagc attcagatgt ctggttgaag atgctggaga
tgttgccttc 1620attaaacaca ctattgtacc agagatgact gatggtagtg
gtccagtttg ggcacagaac 1680ctgatgtcct ctgactttga actactctgc
caggatggta ctaccaagcc agtcacacat 1740ttccgtgagt gccacctggc
caaggtgccc gcccatgctg tgataacacg cccagagtcc 1800cgtggagagg
ttgtgtccat ccttctggag cagcaggcca ggttcggctc aagcggcagc
1860gattcctcat ttaatatgtt caagccagat tttggaaaga acttgctctt
caaggattcc 1920acaaagtgtc tccaggagat cccaagcggc accaaattcc
agggtttcct tggggaagag 1980tacatgatcg ccatgcaatc gctcagggag
tgctccaaca gtacctcaga tttggagaag 2040gcatgcactt tccattcctg c
206133687PRTArtificial SequenceSerotransferrin 33Met Lys Leu Leu
Leu Val Ser Ala Leu Leu Gly Cys Phe Ala Thr Val1 5 10 15Tyr Ala Ala
Pro Ala Glu Gly Met Val Arg Trp Cys Val Lys Ser Glu 20 25 30Lys Glu
Leu Lys Lys Cys His Asp Leu Ala Ala Asn Val Ala Gly Phe 35 40 45Ser
Cys Val Arg Arg Asp Asp Ser Leu Glu Cys Ile Gln Ala Ile Lys 50 55
60Arg Glu Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Asp Ile Tyr Ile65
70 75 80Ala Gly Leu His Asn Tyr Asn Leu Gln Pro Ile Ile Ala Glu Asp
Tyr 85 90 95Gly Glu Asp Ser Asp Thr Cys Tyr Tyr Ala Val Ala Val Ala
Lys Lys 100 105 110Gly Thr Glu Phe Gly Phe Leu Asp Leu Arg Gly Lys
Lys Ser Cys His 115 120 125Thr Gly Leu Gly Lys Ser Ala Gly Trp Asn
Ile Pro Ile Gly Thr Leu 130 135 140Val Thr Val Gly Gln Ile Gln Trp
Ala Gly Ile Glu Asp Arg Pro Val145 150 155 160Glu Ser Ala Val Ser
Asp Phe Phe Asn Ala Ser Cys Ala Pro Gly Ala 165 170 175Asn Arg Asp
Ser Gln Leu Cys Gln Leu Cys Met Gly Asp Cys Ser Arg 180 185 190Ser
His Asn Glu Pro Tyr Tyr Asp Tyr Ser Gly Ala Phe Gln Cys Leu 195 200
205Lys Asp Gly Ala Gly Glu Val Ala Phe Ile Lys His Leu Thr Val Pro
210 215 220Ala Ala Glu Lys Ala Ser Tyr Glu Leu Leu Cys Lys Asp Asn
Thr Arg225 230 235 240Ala Pro Ile Asp Ser Tyr Lys Thr Cys His Leu
Ala Arg Val Pro Ala 245 250 255His Ala Val Val Ser Arg Lys Asp Pro
Arg Leu Ala Asn Leu Ile Tyr 260 265 270Ser Lys Leu Met Ala Val Thr
Asn Phe Asn Leu Phe Ser Ser Asp Gly 275 280 285Tyr Ala Ala Lys Asn
Leu Met Phe Lys Asp Ser Thr Gln Asn Leu Val 290 295 300Gln Leu Pro
Met Thr Thr Asp Ser Phe Leu Tyr Leu Gly Ala Glu Tyr305 310 315
320Met Ser Thr Ile Arg Ser Leu Thr Lys Ala Gln Ala Thr Gly Val Thr
325 330 335Ser Arg Ala Ile Lys Trp Cys Ala Val Gly His Lys Glu Lys
Val Lys 340 345 350Cys Asp Ala Trp Thr Ile Asn Ser Phe Thr Asp Gly
Asp Ser Arg Ile 355 360 365Glu Cys Gln Asp Ala Pro Thr Val Asp Glu
Cys Ile Lys Lys Ile Met 370 375 380Arg Lys Glu Ala Asp Ala Ile Ala
Val Asp Gly Gly Glu Val Phe Thr385 390 395 400Ala Gly Lys Cys Gly
Leu Val Pro Val Met Val Glu Gln Tyr Asp Glu 405 410 415Val Arg Cys
Ser Ala Pro Gly Glu Ala Ser Ser Tyr Phe Ala Val Ala 420 425 430Val
Ala Lys Arg Gly Ser Gly Leu Thr Trp Thr Thr Leu Lys Gly Lys 435 440
445Arg Ser Cys His Thr Gly Leu Gly Arg Thr Ala Gly Trp Asn Ile Pro
450 455 460Met Gly Leu Ile His Arg Arg Thr Met Asn Cys Asp Phe Thr
Thr Tyr465 470 475 480Phe Ser Lys Gly Cys Ala Pro Gly Phe Glu Val
Asp Ser Pro Phe Cys 485 490 495Ala Gln Cys Lys Gly Ser Gly Lys Ser
Val Gly Gly Asp Gly Ser Lys 500 505 510Cys Lys Ala Ser Ser Glu Glu
Gln Tyr Tyr Gly Tyr Asn Gly Ala Phe 515 520 525Arg Cys Leu Val Glu
Asp Ala Gly Asp Val Ala Phe Ile Lys His Thr 530 535 540Ile Val Pro
Glu Met Thr Asp Gly Ser Gly Pro Val Trp Ala Gln Asn545 550 555
560Leu Met Ser Ser Asp Phe Glu Leu Leu Cys Gln Asp Gly Thr Thr Lys
565 570 575Pro Val Thr His Phe Arg Glu Cys His Leu Ala Lys Val Pro
Ala His 580 585 590Ala Val Ile Thr Arg Pro Glu Ser Arg Gly Glu Val
Val Ser Ile Leu 595 600 605Leu Glu Gln Gln Ala Arg Phe Gly Ser Ser
Gly Ser Asp Ser Ser Phe 610 615 620Asn Met Phe Lys Pro Asp Phe Gly
Lys Asn Leu Leu Phe Lys Asp Ser625 630 635 640Thr Lys Cys Leu Gln
Glu Ile Pro Ser Gly Thr Lys Phe Gln Gly Phe 645 650 655Leu Gly Glu
Glu Tyr Met Ile Ala Met Gln Ser Leu Arg Glu Cys Ser 660 665 670Asn
Ser Thr Ser Asp Leu Glu Lys Ala Cys Thr Phe His Ser Cys 675 680
6853412PRTArtificial SequenceSerotransferrin fragment 34Ala Gln Ala
Thr Gly Val Thr Ser Arg Ala Ile Lys1 5 103510PRTArtificial
SequenceSerotransferrin fragment 35Ala Thr Gly Val Thr Ser Arg Ala
Ile Lys1 5 103611PRTArtificial SequenceSerotransferrin fragment
36Asp Pro Arg Leu Ala Asn Leu Ile Tyr Ser Lys1 5
103712PRTArtificial SequenceSerotransferrin fragment 37Gly Thr Glu
Phe Gly Phe Leu Asp Leu Arg Gly Lys1 5 103813PRTArtificial
SequenceSerotransferrin fragment 38Lys Gly Thr Glu Phe Gly Phe Leu
Asp Leu Arg Gly Lys1 5 103914PRTArtificial SequenceSerotransferrin
fragment 39Lys Gly Thr Glu Phe Gly Phe Leu Asp Leu Arg Gly Lys Lys1
5 104018PRTArtificial SequenceSerotransferrin fragment 40Leu Met
Ala Val Thr Asn Phe Asn Leu Phe Ser Ser Asp Gly Tyr Ala1 5 10 15Ala
Lys4111PRTArtificial SequenceSerotransferrin fragment 41Arg Gly Ser
Gly Leu Thr Trp Thr Thr Leu Lys1 5 10421518DNAArtificial
SequenceMyosin binding protein H-like 42atgcctgcga agcctgcccc
tatcaagaag gcagccccga aagagcctgc tcctaagaag 60gaggaagcac ctgcacctgc
acccgcggag gcggctcctg ctgaagttgc tccccctgcc 120gaggtcgaag
ctccaccggc agaggctgcc cctgccgaag cagctgcccc tgctgaggca
180gctccaaccg aagaaaaggc tcccgcagaa cccgcgcctg agcagcccaa
agcacccaca 240ccccctcctc cagatgagcc caccagtgag cccctggaag
tgacagtgga cgacaagagc 300gacacttcag tgaccatcac ctggaggcct
ccaaagacta ttccagccaa ctctggcctg 360gatggataca ctgtggaaat
caccaaggag ggaactgaag actggaaagc agccaacgag 420gatctgaccg
tgtcctgccg ttatgtcatc aagaaccaga ctaccggcga ccgtctgctg
480atccgagtgg ttgctgtaaa ccctggtggc cgcagccccc ctgctaccat
cgccgacccc 540gttctggtca aggaggttgg ggctcgcccc aaagtccgcc
tccctcgttt cctgaggcag 600aggtatgtca agaaagtcgg cgagaagatc
aacattgtca tcccgtactc tggcaaacct 660aagccagtgg ttagctggtt
aaaggatgga cagcccctcg actcaaagag ggccaacatc 720cgtacatctg
acagagacag catcctgttc atccgtcaag cagagagggt ggactctggc
780tcatacgaaa tgtgcgtgaa ggtggacgac tttgaggaca aagctgctat
catcatccag 840attattgagt tacccgggcc accagccagc attaagattg
tggatgtctg gggattcaac 900gttgctttgg agtggaccgt acccaaagat
aatggaaaca cagagatcac aggctacact 960gtccagaaag ctgacaagaa
gaccggggac tggttcaaca ttttggagca ctacgccaga 1020ctaaatgcca
ccatctcaga cctcattatg ggcaacacct acaccttcag ggtctttgca
1080gagaacaagt gcggactcag tgaggaatgt gccgtgacca agggagaagc
caccattgtg 1140aaggaggtta ttgactacaa acctaccccg ttcgtggagc
atgacttcac cgaggctccc 1200aagttcacca ctgttctgaa cgacaggtcc
accactgttg gctacagcac caagctgctg 1260tgttctgtga ggggatgccc
caagcctaag attatgtgga tgaagaacaa gatgattctc 1320aacaacatgg
acaaccccaa gtacaggatg atcagcacag ggggcatctg caccctggag
1380atccgtaagc ctggcccata cgacggtggt gagtatgtct gcagagccga
gaatacactg 1440gggaaggtcg acaccggctg caagctggag gtcagaaagc
ctgggcaagc agatgctgac 1500aaagacaaga aggaacag
151843506PRTArtificial SequenceMyosin binding protein H-like 43Met
Pro Ala Lys Pro Ala Pro Ile Lys Lys Ala Ala Pro Lys Glu Pro1 5 10
15Ala Pro Lys Lys Glu Glu Ala Pro Ala Pro Ala Pro Ala Glu Ala Ala
20 25 30Pro Ala Glu Val Ala Pro Pro Ala Glu Val Glu Ala Pro Pro Ala
Glu 35 40 45Ala Ala Pro Ala Glu Ala Ala Ala Pro Ala Glu Ala Ala Pro
Thr Glu 50 55 60Glu Lys Ala Pro Ala Glu Pro Ala Pro Glu Gln Pro Lys
Ala Pro Thr65 70 75 80Pro Pro Pro Pro Asp Glu Pro Thr Ser Glu Pro
Leu Glu Val Thr Val 85 90 95Asp Asp Lys Ser Asp Thr Ser Val Thr Ile
Thr Trp Arg Pro Pro Lys 100 105 110Thr Ile Pro Ala Asn Ser Gly Leu
Asp Gly Tyr Thr Val Glu Ile Thr 115 120 125Lys Glu Gly Thr Glu Asp
Trp Lys Ala Ala Asn Glu Asp Leu Thr Val 130 135 140Ser Cys Arg Tyr
Val Ile Lys Asn Gln Thr Thr Gly Asp Arg Leu Leu145 150 155 160Ile
Arg Val Val Ala Val Asn Pro Gly Gly Arg Ser Pro Pro Ala Thr 165 170
175Ile Ala Asp Pro Val Leu Val Lys Glu Val Gly Ala Arg Pro Lys Val
180 185 190Arg Leu Pro Arg Phe Leu Arg Gln Arg Tyr Val Lys Lys Val
Gly Glu 195 200 205Lys Ile Asn Ile Val Ile Pro Tyr Ser Gly Lys Pro
Lys Pro Val Val 210 215 220Ser Trp Leu Lys Asp Gly Gln Pro Leu Asp
Ser Lys Arg Ala Asn Ile225 230 235 240Arg Thr Ser Asp Arg Asp Ser
Ile Leu Phe Ile Arg Gln Ala Glu Arg 245 250 255Val Asp Ser Gly Ser
Tyr Glu Met Cys Val Lys Val Asp Asp Phe Glu 260 265 270Asp Lys Ala
Ala Ile Ile Ile Gln Ile Ile Glu Leu Pro Gly Pro Pro 275 280 285Ala
Ser Ile Lys Ile Val Asp Val Trp Gly Phe Asn Val Ala Leu Glu 290 295
300Trp Thr Val Pro Lys Asp Asn Gly Asn Thr Glu Ile Thr Gly Tyr
Thr305 310 315 320Val Gln Lys Ala Asp Lys Lys Thr Gly Asp Trp Phe
Asn Ile Leu Glu 325 330 335His Tyr Ala Arg Leu Asn Ala Thr Ile Ser
Asp Leu Ile Met Gly Asn 340 345 350Thr Tyr Thr Phe Arg Val Phe Ala
Glu Asn Lys Cys Gly Leu Ser Glu 355 360 365Glu Cys Ala Val Thr Lys
Gly Glu Ala Thr Ile Val Lys Glu Val Ile 370 375 380Asp Tyr Lys Pro
Thr Pro Phe Val Glu His Asp Phe Thr Glu Ala Pro385 390 395 400Lys
Phe Thr Thr Val Leu Asn Asp Arg Ser Thr Thr Val Gly Tyr Ser 405 410
415Thr Lys Leu Leu Cys Ser Val Arg Gly Cys Pro Lys Pro Lys Ile Met
420 425 430Trp Met Lys Asn Lys Met Ile Leu Asn Asn Met Asp Asn Pro
Lys Tyr 435 440 445Arg Met Ile Ser Thr Gly Gly Ile Cys Thr Leu Glu
Ile Arg Lys Pro 450 455 460Gly Pro Tyr Asp Gly Gly Glu Tyr Val Cys
Arg Ala Glu Asn Thr Leu465 470 475 480Gly Lys Val Asp Thr Gly Cys
Lys Leu Glu Val Arg Lys Pro Gly Gln 485 490 495Ala Asp Ala Asp Lys
Asp Lys Lys Glu Gln 500 5054418PRTArtificial SequenceMyosin binding
protein H-like fragment 44Ala Ala Ile Ile Ile Gln Ile Ile Glu Leu
Pro Gly Pro Pro Ala Ser1 5 10 15Ile Lys4518PRTArtificial
SequenceMyosin binding protein H-like fragment 45Ala Ala Pro Ala
Glu Ala Ala Ala Pro Ala Glu Ala Ala Pro Thr Glu1 5 10 15Glu
Lys4611PRTArtificial SequenceMyosin binding protein H-like fragment
46Ala Pro Ala Glu Pro Ala Pro Glu Gln Pro Lys1 5
104714PRTArtificial SequenceMyosin binding protein H-like fragment
47Asp Asn Gly Asn Thr Glu Ile Thr Gly Tyr Thr Val Gln Lys1 5
104820PRTArtificial SequenceMyosin binding protein H-like fragment
48Glu Val Ile Asp Tyr Lys Pro Thr Pro Phe Val Glu His Asp Phe Thr1
5 10 15Glu Ala Pro Lys 204917PRTArtificial SequenceMyosin binding
protein H-like fragment 49Phe Thr Thr Val Leu Asn Asp Arg Ser Thr
Thr Val Gly Tyr Ser Thr1 5 10 15Lys5010PRTArtificial SequenceMyosin
binding protein H-like fragment 50Ile Asn Ile Val Ile Pro Tyr Ser
Gly Lys1 5 105119PRTArtificial SequenceMyosin binding protein
H-like fragment 51Ile Asn Ile Val Ile Pro Tyr Ser Gly Lys Pro Lys
Pro Val Val Ser1 5 10 15Trp Leu Lys5233PRTArtificial SequenceMyosin
binding protein H-like fragment 52Asn Gln Thr Thr Gly Asp Arg Leu
Leu Ile Arg Val Val Ala Val Asn1 5 10 15Pro Gly Gly Arg Ser Pro Pro
Ala Thr Ile Ala Asp Pro Val Leu Val 20 25 30Lys5313PRTArtificial
SequenceMyosin binding protein H-like fragment 53Ser Asp Thr Ser
Val Thr Ile Thr Trp Arg Pro Pro Lys1 5 105417PRTArtificial
SequenceMyosin binding protein H-like fragment 54Thr Ile Pro Ala
Asn Ser Gly Leu Asp Gly Tyr Thr Val Glu Ile Thr1 5 10
15Lys551377DNAArtificial SequenceDesmin (fragment) 55atttcctctt
accgccgcac tttcggttct ggtattggat ccaccccagg catgtcctcc 60atgttctccc
acggtggacg cggctcctcc ggctcggccc acatgtcctc cagagtctac
120gagatgacca aaagctccgc ccgccccagc tactcctccg gcagtattcg
ctcctcctcc 180ggcggcgcga tgcgctcgta cgccgggatg ggcgagaagc
tggacttcaa cctggccgac 240gccactaacc gcgacttcct ggacaccaga
accaatgaga aggcagagtt gcagcacctg 300aacgaccggt tcgccagcta
catcgagaag gtccgtttcc tggagcagca gaacgctact 360ctggtggtgg
agatcgagag gctgaggggt cacgagccga cccgcgtggc cgagatgtac
420gaggaggaga tgagagagct gaggcgtcag gtggacggca tgtccaatga
ccgagcccgc 480atggaggtgg agagagacaa cttggctgac gacctgcaga
aactcaaact cagactgcag 540gaggtgatcc accagaggga agaggcagag
aacaacctgt ctgccttcag agctgacgtg 600gactctgcca cgctggccag
gctggacctg gagagacgca ttgaaagcct gcaggaggag 660atcaccttcc
tcaagaagat ccacgaggag gagatccatg agctgacgag ccagatgcag
720gagacctcgg tgcaggtcca gatggacatg tccaaaccag acctgaccgt
tgccctcagg 780gacatccgta tgcagtacga gggtatcgca gccaagaaca
tctctgaggc agaggactgg 840tacaagtcca aggtgtcaga cctgaaccag
gctgtgaaca agaacaacga tgctctgaga 900caggccaaac aggagagcat
ggagttcagg catcagatcc agtcctacac ctgtgagatc 960gactctctca
agggaaccaa cgagtctctg ctgaggcaga tgagggagat ggaggatcgt
1020ctgggaaacg aggctggagg ttaccaggac tccgtcaccc gtctggaggc
tgagatcgcc 1080aagatgaagg atgagatggc tcgtcacctc agagagtacc
aggacctcct caatgtcaag 1140atggccctgg atatagagat cgctacctac
aggaagctac tggagggaga ggagagccga 1200atcactgtat ctggctccaa
gtcttctcac tctggctccc actctgctgc ctcattatat 1260tccactgttg
gattcagaga gaccagccca gacgtcggac gatcagcaga ggtccactcc
1320aagaagactg tcctgatcaa gaccatcgag acccgcgatg gagaggtcgt cagcgag
137756459PRTArtificial SequenceDesmin (fragment) 56Ile Ser Ser Tyr
Arg Arg Thr Phe Gly Ser Gly Ile Gly Ser Thr Pro1 5 10 15Gly Met Ser
Ser Met Phe Ser His Gly Gly Arg Gly Ser Ser Gly Ser 20 25 30Ala His
Met Ser Ser Arg Val Tyr Glu Met Thr Lys Ser Ser Ala Arg 35 40 45Pro
Ser Tyr Ser Ser Gly Ser Ile Arg Ser Ser Ser Gly Gly Ala Met 50 55
60Arg Ser Tyr Ala Gly Met Gly Glu Lys Leu Asp Phe Asn Leu Ala Asp65
70 75 80Ala Thr Asn Arg Asp Phe Leu Asp Thr Arg Thr Asn Glu Lys Ala
Glu 85 90 95Leu Gln His Leu Asn Asp Arg Phe Ala Ser Tyr Ile Glu Lys
Val Arg 100 105 110Phe Leu Glu Gln Gln Asn Ala Thr Leu Val Val Glu
Ile Glu Arg Leu 115 120 125Arg Gly His Glu Pro Thr Arg
Val Ala Glu Met Tyr Glu Glu Glu Met 130 135 140Arg Glu Leu Arg Arg
Gln Val Asp Gly Met Ser Asn Asp Arg Ala Arg145 150 155 160Met Glu
Val Glu Arg Asp Asn Leu Ala Asp Asp Leu Gln Lys Leu Lys 165 170
175Leu Arg Leu Gln Glu Val Ile His Gln Arg Glu Glu Ala Glu Asn Asn
180 185 190Leu Ser Ala Phe Arg Ala Asp Val Asp Ser Ala Thr Leu Ala
Arg Leu 195 200 205Asp Leu Glu Arg Arg Ile Glu Ser Leu Gln Glu Glu
Ile Thr Phe Leu 210 215 220Lys Lys Ile His Glu Glu Glu Ile His Glu
Leu Thr Ser Gln Met Gln225 230 235 240Glu Thr Ser Val Gln Val Gln
Met Asp Met Ser Lys Pro Asp Leu Thr 245 250 255Val Ala Leu Arg Asp
Ile Arg Met Gln Tyr Glu Gly Ile Ala Ala Lys 260 265 270Asn Ile Ser
Glu Ala Glu Asp Trp Tyr Lys Ser Lys Val Ser Asp Leu 275 280 285Asn
Gln Ala Val Asn Lys Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln 290 295
300Glu Ser Met Glu Phe Arg His Gln Ile Gln Ser Tyr Thr Cys Glu
Ile305 310 315 320Asp Ser Leu Lys Gly Thr Asn Glu Ser Leu Leu Arg
Gln Met Arg Glu 325 330 335Met Glu Asp Arg Leu Gly Asn Glu Ala Gly
Gly Tyr Gln Asp Ser Val 340 345 350Thr Arg Leu Glu Ala Glu Ile Ala
Lys Met Lys Asp Glu Met Ala Arg 355 360 365His Leu Arg Glu Tyr Gln
Asp Leu Leu Asn Val Lys Met Ala Leu Asp 370 375 380Ile Glu Ile Ala
Thr Tyr Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg385 390 395 400Ile
Thr Val Ser Gly Ser Lys Ser Ser His Ser Gly Ser His Ser Ala 405 410
415Ala Ser Leu Tyr Ser Thr Val Gly Phe Arg Glu Thr Ser Pro Asp Val
420 425 430Gly Arg Ser Ala Glu Val His Ser Lys Lys Thr Val Leu Ile
Lys Thr 435 440 445Ile Glu Thr Arg Asp Gly Glu Val Val Ser Glu 450
4555717PRTArtificial SequenceA part of Desmin (fragment) 57Asp Glu
Met Ala Arg His Leu Arg Glu Tyr Gln Asp Leu Leu Asn Val1 5 10
15Lys5821PRTArtificial SequenceA part of Desmin (fragment) 58Leu
Asp Phe Asn Leu Ala Asp Ala Thr Asn Arg Asp Phe Leu Asp Thr1 5 10
15Arg Thr Asn Glu Lys 205910PRTArtificial SequenceA part of Desmin
(fragment) 59Asn Ile Ser Glu Ala Glu Asp Trp Tyr Lys1 5
1060822DNAArtificial SequenceCapping protein (Actin filament)
muscle Z-line beta 60atgaatgacc agcagctgga ctgtgctctg gacctgatga
ggcgtcttcc tccccagcag 60atcgagaaga atctcagtga cctcatcgac ctggtgccca
gtctgtgtga ggacctactc 120tcctctgtgg accagcctct gaagatcgcc
cgggacaagg tagtggggaa agactacctg 180ctctgtgact ataacagaga
cggtgactcc tacagatccc catggagtaa taagtatgag 240ccacccatcg
acgatggtgc catgccatct gcccgcctgc gcaaactaga ggtggaagcc
300aacaatgcct ttgaccagta tagagacctg tactttgagg gtggcgtatc
gtctgtgtat 360ctgtgggact tggatcatgg ctttgctggg gtcatcctca
tcaagaaggc tggagacggc 420tccaagaaga tcaaaggctg ctgggactcc
atccatgtgg tggaggtgca ggagaagtcc 480agcggacgga ccgctcacta
caaactcacc tccaccgtca tgctgtggct ccagacgacc 540aaggccgggt
ctggaaccat gaacctgggt ggcagtctga caagacagat ggagaaagac
600gagacagttg gagagtcttc cccacatatt gccaacatcg gccgcctggt
ggaggatatg 660gagaataaga ttcgctccac tctcaacgag atctactttg
ggaagaccaa ggacatcgtc 720aatggtttaa gatctattga ctctctgcct
gataaccaaa agtaccggca gctccagaag 780gagctgtctc aggtccttac
ccagcgccag atcttcattg ac 82261274PRTArtificial SequenceCapping
protein (Actin filament) muscle Z-line beta 61Met Asn Asp Gln Gln
Leu Asp Cys Ala Leu Asp Leu Met Arg Arg Leu1 5 10 15Pro Pro Gln Gln
Ile Glu Lys Asn Leu Ser Asp Leu Ile Asp Leu Val 20 25 30Pro Ser Leu
Cys Glu Asp Leu Leu Ser Ser Val Asp Gln Pro Leu Lys 35 40 45Ile Ala
Arg Asp Lys Val Val Gly Lys Asp Tyr Leu Leu Cys Asp Tyr 50 55 60Asn
Arg Asp Gly Asp Ser Tyr Arg Ser Pro Trp Ser Asn Lys Tyr Glu65 70 75
80Pro Pro Ile Asp Asp Gly Ala Met Pro Ser Ala Arg Leu Arg Lys Leu
85 90 95Glu Val Glu Ala Asn Asn Ala Phe Asp Gln Tyr Arg Asp Leu Tyr
Phe 100 105 110Glu Gly Gly Val Ser Ser Val Tyr Leu Trp Asp Leu Asp
His Gly Phe 115 120 125Ala Gly Val Ile Leu Ile Lys Lys Ala Gly Asp
Gly Ser Lys Lys Ile 130 135 140Lys Gly Cys Trp Asp Ser Ile His Val
Val Glu Val Gln Glu Lys Ser145 150 155 160Ser Gly Arg Thr Ala His
Tyr Lys Leu Thr Ser Thr Val Met Leu Trp 165 170 175Leu Gln Thr Thr
Lys Ala Gly Ser Gly Thr Met Asn Leu Gly Gly Ser 180 185 190Leu Thr
Arg Gln Met Glu Lys Asp Glu Thr Val Gly Glu Ser Ser Pro 195 200
205His Ile Ala Asn Ile Gly Arg Leu Val Glu Asp Met Glu Asn Lys Ile
210 215 220Arg Ser Thr Leu Asn Glu Ile Tyr Phe Gly Lys Thr Lys Asp
Ile Val225 230 235 240Asn Gly Leu Arg Ser Ile Asp Ser Leu Pro Asp
Asn Gln Lys Tyr Arg 245 250 255Gln Leu Gln Lys Glu Leu Ser Gln Val
Leu Thr Gln Arg Gln Ile Phe 260 265 270Ile Asp6224PRTArtificial
SequenceCapping protein (Actin filament) muscle Z-line beta
fragment 62Asp Glu Thr Val Gly Glu Ser Ser Pro His Ile Ala Asn Ile
Gly Arg1 5 10 15Leu Val Glu Asp Met Glu Asn Lys 206317PRTArtificial
SequenceCapping protein (Actin filament) muscle Z-line beta
fragment 63Asp Ile Val Asn Gly Leu Arg Ser Ile Asp Ser Leu Pro Asp
Asn Gln1 5 10 15Lys6414PRTArtificial SequenceCapping protein (Actin
filament) muscle Z-line beta fragment 64Glu Leu Ser Gln Val Leu Thr
Gln Arg Gln Ile Phe Ile Asp1 5 106512PRTArtificial SequenceCapping
protein (Actin filament) muscle Z-line beta fragment 65Ile Arg Ser
Thr Leu Asn Glu Ile Tyr Phe Gly Lys1 5 106613PRTArtificial
SequenceCapping protein (Actin filament) muscle Z-line beta
fragment 66Leu Thr Ser Thr Val Met Leu Trp Leu Gln Thr Thr Lys1 5
106710PRTArtificial SequenceCapping protein (Actin filament) muscle
Z-line beta fragment 67Ser Ile Asp Ser Leu Pro Asp Asn Gln Lys1 5
106817PRTArtificial SequenceCapping protein (Actin filament) muscle
Z-line beta fragment 68Tyr Glu Pro Pro Ile Asp Asp Gly Ala Met Pro
Ser Ala Arg Leu Arg1 5 10 15Lys695799DNAArtificial Sequencemyosin
heavy chain, fast skeletal muscle-like 69atgagtacgg acgcggagat
gcaagtctac ggcaaggctg ccatatacct tcgtaagtct 60gagaaggaga ggatggaggc
acaagccatg ccctttgatt caaagaacgc ctgctatgtg 120acagacaagg
tggagctgta ccttaagggt ttagtcactg ccagggccga cgggaagtgt
180actgtgacag ctttagactt tcgtgaagga aaagagttca aagatgcaga
catctatgag 240atgaaccccc ctaagtacga caagattgag gacatggcca
tgatgaccta cctgaatgaa 300gcctctgtgt tgtataacct caaagagcgt
tatgcagcat ggatgatcta tacctactct 360gggctcttct gtgccacggt
gaacccctac aagtggctcc cagtgtacga cgaagaggtt 420gtcaacgcct
acagagggaa gaagagggtg gaggctccac cacatatctt ctccgtctct
480gacaacgcct ttcagttcat gatgattgat aaggagaacc agtccgtcct
gattactgga 540gaatccggtg caggaaagac tgtcaacacc aagcgtgtca
tccagtactt tgccaccatt 600gcagtgtctg gtggcaagaa ggaagcagac
cccaacaaaa tgcaggggtc tcttgaggat 660cagatcattg cagctaaccc
tctgctagag tcttacggta atgccaagac agtgaggaac 720gacaactcgt
ctcgctttgg taaattcatc aggattcact tccaagctgg taaactggct
780aaagctgaca ttgagaccta cctgctggag aagtccagag tgtccttcca
actgcccgat 840gagagaggct accacatctt cttccagatg atgacaggcc
acaaacctga gctagttgaa 900ttggcactcc tcaccaccaa cccctacgac
ttccccatgt gcagccaggg tcagattgct 960gtggccagca tcaacgacaa
tgaagagctg gatgccacag atgaagccat tacaatcctg 1020ggcttcacta
atgaggagaa gcttggaata tacaagctga caggagctgt agtgcaccat
1080ggcaacttga aattcaagca gaagcagcgt gaggagcagg ccgagccaga
cggcacagag 1140gtggctgata aaatcgccta cctgctgggc ctgaactcag
ctgagatgtt gaaagctctg 1200tgctacccca gagtgaaggt cggcaacgag
tatgtgacca agggacagac tgtggctcag 1260gttaataact cagtcagtgc
tctggccaag tccatctatg agaggatgtt cttgtggatg 1320gtcatccgta
tcaatgagat gttggacacc aagaatccaa ggcagttcta tatcggtgtg
1380cttgacattg ccgggtttga gatctttgat tacaacagca tggagcagct
gtgcatcaac 1440ttcaccaatg agaaactgca acagtttttc aaccacacca
tgttcgtcct ggagcaagag 1500gagtacaaga aggagggaat cgtctgggcc
ttcattgact tcggcatgga tttggctgcc 1560tgcattgagc ttattgagaa
gccattgggc atcttctcca tccttgaaga ggagtgcatg 1620ttccccaagt
cttcagacac taccttcaag gacaagctgt acgcccagca tcttggtaaa
1680acaaaggcgt ttgagaagcc caagcctgcc aaaggcaagg cagaggccca
cttctccctg 1740gtgcattacg ccggtactgt ggactacaac atcactggct
ggctggagaa gaacaaggac 1800cccttgaacg actcagtttg tcaactgtat
gggaagtccg gagtcaaaat tttggctgcc 1860ctgtatcccc ctccccctcc
tgaggataaa gccaagaaag gaggcaagaa gaagggtggt 1920tccatgcaga
ctgtgtcctc ccagttcagg gagaacttac ataagctgat gaccaacttg
1980aggagcactc atcctcactt tgtgcgctgc ctgatcccca acgagtcaaa
gactccaggt 2040ctgatggaga acttcctggt tatccaccag ctcaggtgta
atggtgtact ggagggtatc 2100aggatctgca gaaagggctt ccccagcaga
atcatctatg ctgacttcaa gcaaaggtac 2160aaagtactga atgccagcgt
catccctgag ggccagttca tggacaacaa gaaggcttct 2220gagaagctgc
ttggatccat tgatgtgaat cacgaggatt acaagtttgg acacaccaag
2280gtgttcttca aagccggtct gctgggtgtc ctggaggaga tgagagatga
gaagctggcc 2340tctctagtcg gcatgctcca ggctctcagc cgtggattcc
tcatgaggag agagttcacc 2400aagatgatgg agaggagaga atcaatttac
tccatccagt acaacatccg ctcattcatg 2460aatgtgaaaa cctggccatg
gatgaagttg tacttcaaga tcaagcccct gctgcagagc 2520gctgagactg
agaaggagct ggccaacatg aaggagaact atgagaaaat gaagacagac
2580ctggccaagg ctctgtctac aaagaagcaa atggaggaga agttggtgtc
cctgacgcag 2640gagaagaacg acctggcact ccaagtcgca tctgaaggag
agagtctgaa cgatgctgag 2700gaaaggtgcg aggggctcat caagagcaag
atccagcagg aggccaaact caaagagacg 2760accgagaggc tggaggatga
ggaggagatc aatgctgagt tgactgccaa gaagaggaag 2820ctggaggatg
agtgctctga gctgaagaag gacattgatg atctggagct caccctggcc
2880aaagtggaga aggagaagca cgccactgaa aacaaggtta aaaacctgac
agaggagatg 2940gcgtctatgg atgagagtgt tgccaagctg accaaggaga
agaaagccct acaagaggcc 3000caccagcaga cactggatga cctgcaggca
gaggaggaca aagtcaacac tctgaccaag 3060gccaagacca agctggaaca
gcaagtggac gaccttgagg gttctctgga gcaagagaag 3120aagctccgca
tggaccttga gagatccaag agaaagctgg agggagatct gaaactggcc
3180caggagtcca taatggacct ggagaatgac aagcagcaag ctgatgagaa
aatcaagaag 3240aaggagtttg agaccactca gctcctcagc aaggttgagg
atgagcagtc tctgggagct 3300cagctgcaga agaagatcaa ggaactccag
gcccgtattg aggaactgga ggaggaaatt 3360gaggctgagc gtgctgccag
ggctaaggtt gagaagcaga gggctgatct ctccagggaa 3420cttgaggaga
tcagcgagag gctggaggag gccggaggcg ccactgctgc tcagattgag
3480atgaacaaga agcgtgaggc tgagttccag aagctgcgtc gtgatcttga
agagtccacc 3540ctgcagcatg aggccacagc cgccgctctg cgcaagaagc
aggccgacag tgtggctgag 3600ctcggggagc agatcgacaa cctgcagcgc
gtcaagcaga agctggagaa ggagaagagc 3660gagtacaaga tggagattga
tgacctctcc agcaacatgg aggccgttgc caaggctaag 3720ggcaatctag
agaagatgtg ccgtactctt gaggaccagc tgagcgagct caagactaag
3780aatgatgaga atgttcgcca ggtcaacgac atcagcggac agagggccag
actcctgaca 3840gaaaatggtg agtttggtag gcagctggag gagaaggaag
ccctggtgtc tcagctgacc 3900agaggcaaac aggccttcac ccagcaggtg
gaggagctga agagggcgat tgaggaggag 3960gtcaaggcta aaaatgcact
ggcccacgga gttcagtctg cccgccatga ctgtgacctc 4020ctgagggagc
agtttgagga ggagcaggag gccaaggcag agctgcaacg cggcatgtcc
4080aaggccaata gtgaggtggc tcagtggagg actaagtatg aaactgatgc
catccagcgc 4140acagaggagc tggaggaggc caagaagaag ctggcccagc
gtctgcagga tgccgaggag 4200accattgagg cgaccaactc caagtgcgcc
tccctggaga agaccaagca gagactacag 4260ggagaggtgg aggacctcat
gattgatgtt gagagagcca acgcattggc cgccaacctc 4320gacaagaagc
agaggaactt tgacaaggtt ctggcagagt ggaagcagaa gtatgaggag
4380ggtcaggctg agctggaagg agctcagaag gaggctcgct ctatgagcac
tgagctcttc 4440aagatgaaga actcctacga ggaggctctg gatcatctgg
agactctgaa gagagagaac 4500aagaacctgc aacaggagat ctctgacctt
actgagcaga ttggagagac tggcaagagc 4560atccatgagc tggagaaggc
caagaagacc gtggagacag agaagtctga gatccagacc 4620gctctggagg
aggctgaggg cacactggag cacgaggaat ccaagattct gcgtgtgcag
4680ctggagctga accagatcaa gggtgaggtg gacaggaaga tcgctgagaa
ggacgaggag 4740atggagcaga tcaagaggaa cagccagagg atggttgact
ccatgcagag caccctggac 4800tctgaggtca ggagcaggaa tgatgccctg
agggtgaaga agaagatgga gggagacctg 4860aacgagatgg agatccagct
gagccactcc aacaggcagg ccgctgaggc ccagaaacag 4920ctgaggaatg
tccagggaca gctcaaggat gcccaattgc accttgatga tgccgtccgt
4980gtcgcagagg acatgaagga gcaggcagcc atggtggagc gcagaaacgg
tctgatggtg 5040gctgagatcg aggagctgag agttgctctg gagcagacag
agagaggccg caaagtggct 5100gagactgagc tggtagacgc cagcgagcgt
gttggactgc tgcactccca gaacaccagc 5160cttctgaaca ccaagaagaa
gctggagact gacctggtgc aggtgcaggg agaggtggat 5220gacatcgtcc
aggaggccag gaatgcagaa gagaaggcca agaaggcaat cactgatgcg
5280gcaatgatgg ctgaggagct gaagaaggag caggacacca gctctcacct
ggagagaatg 5340aagaagaacc tggagatcac agtcaaggac ctgcagcacc
gcctggatga ggctgagaat 5400ctggccatga agggaggcaa gaaacaactc
cagaaactgg aatccagggt gcgtgagctt 5460gagactgagg tggaggctga
gcagagaaga ggtgtagacg cggtaaaggg agtccgcaag 5520tatgagcgca
gagtcaagga gctcacttac cagactgagg aggataagaa gaatgttaac
5580agacttcagg acctggtaga taagctgcag atgaaagtga aggcctacaa
gaggcacgct 5640gaggaagcgg aggaagcagc aaaccagcac atgtctaagt
tcaggaaggt tcagcatgag 5700ctggaggagg ctgaggagcg tgctgacatc
gctgagactc aggtcaacaa gctcagagcc 5760aagacccgtg actctggaaa
gggaaaagaa gttgctgaa 5799701933PRTArtificial Sequencemyosin heavy
chain, fast skeletal muscle-like 70Met Ser Thr Asp Ala Glu Met Gln
Val Tyr Gly Lys Ala Ala Ile Tyr1 5 10 15Leu Arg Lys Ser Glu Lys Glu
Arg Met Glu Ala Gln Ala Met Pro Phe 20 25 30Asp Ser Lys Asn Ala Cys
Tyr Val Thr Asp Lys Val Glu Leu Tyr Leu 35 40 45Lys Gly Leu Val Thr
Ala Arg Ala Asp Gly Lys Cys Thr Val Thr Ala 50 55 60Leu Asp Phe Arg
Glu Gly Lys Glu Phe Lys Asp Ala Asp Ile Tyr Glu65 70 75 80Met Asn
Pro Pro Lys Tyr Asp Lys Ile Glu Asp Met Ala Met Met Thr 85 90 95Tyr
Leu Asn Glu Ala Ser Val Leu Tyr Asn Leu Lys Glu Arg Tyr Ala 100 105
110Ala Trp Met Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Ala Thr Val Asn
115 120 125Pro Tyr Lys Trp Leu Pro Val Tyr Asp Glu Glu Val Val Asn
Ala Tyr 130 135 140Arg Gly Lys Lys Arg Val Glu Ala Pro Pro His Ile
Phe Ser Val Ser145 150 155 160Asp Asn Ala Phe Gln Phe Met Met Ile
Asp Lys Glu Asn Gln Ser Val 165 170 175Leu Ile Thr Gly Glu Ser Gly
Ala Gly Lys Thr Val Asn Thr Lys Arg 180 185 190Val Ile Gln Tyr Phe
Ala Thr Ile Ala Val Ser Gly Gly Lys Lys Glu 195 200 205Ala Asp Pro
Asn Lys Met Gln Gly Ser Leu Glu Asp Gln Ile Ile Ala 210 215 220Ala
Asn Pro Leu Leu Glu Ser Tyr Gly Asn Ala Lys Thr Val Arg Asn225 230
235 240Asp Asn Ser Ser Arg Phe Gly Lys Phe Ile Arg Ile His Phe Gln
Ala 245 250 255Gly Lys Leu Ala Lys Ala Asp Ile Glu Thr Tyr Leu Leu
Glu Lys Ser 260 265 270Arg Val Ser Phe Gln Leu Pro Asp Glu Arg Gly
Tyr His Ile Phe Phe 275 280 285Gln Met Met Thr Gly His Lys Pro Glu
Leu Val Glu Leu Ala Leu Leu 290 295 300Thr Thr Asn Pro Tyr Asp Phe
Pro Met Cys Ser Gln Gly Gln Ile Ala305 310 315 320Val Ala Ser Ile
Asn Asp Asn Glu Glu Leu Asp Ala Thr Asp Glu Ala 325 330 335Ile Thr
Ile Leu Gly Phe Thr Asn Glu Glu Lys Leu Gly Ile Tyr Lys 340 345
350Leu Thr Gly Ala Val Val His His Gly Asn Leu Lys Phe Lys Gln Lys
355 360 365Gln Arg Glu Glu Gln Ala Glu Pro Asp Gly Thr Glu Val Ala
Asp Lys 370 375 380Ile Ala Tyr Leu Leu Gly Leu Asn Ser Ala Glu Met
Leu Lys Ala Leu385 390 395 400Cys Tyr Pro Arg Val Lys Val Gly Asn
Glu Tyr Val Thr Lys Gly Gln 405 410 415Thr Val Ala Gln Val Asn Asn
Ser Val Ser Ala Leu Ala Lys Ser Ile 420 425 430Tyr Glu Arg Met Phe
Leu Trp Met Val Ile Arg Ile Asn Glu Met Leu 435 440 445Asp Thr Lys
Asn Pro Arg Gln Phe Tyr Ile Gly Val Leu Asp Ile Ala 450 455 460Gly
Phe Glu Ile Phe Asp Tyr Asn Ser Met Glu Gln
Leu Cys Ile Asn465 470 475 480Phe Thr Asn Glu Lys Leu Gln Gln Phe
Phe Asn His Thr Met Phe Val 485 490 495Leu Glu Gln Glu Glu Tyr Lys
Lys Glu Gly Ile Val Trp Ala Phe Ile 500 505 510Asp Phe Gly Met Asp
Leu Ala Ala Cys Ile Glu Leu Ile Glu Lys Pro 515 520 525Leu Gly Ile
Phe Ser Ile Leu Glu Glu Glu Cys Met Phe Pro Lys Ser 530 535 540Ser
Asp Thr Thr Phe Lys Asp Lys Leu Tyr Ala Gln His Leu Gly Lys545 550
555 560Thr Lys Ala Phe Glu Lys Pro Lys Pro Ala Lys Gly Lys Ala Glu
Ala 565 570 575His Phe Ser Leu Val His Tyr Ala Gly Thr Val Asp Tyr
Asn Ile Thr 580 585 590Gly Trp Leu Glu Lys Asn Lys Asp Pro Leu Asn
Asp Ser Val Cys Gln 595 600 605Leu Tyr Gly Lys Ser Gly Val Lys Ile
Leu Ala Ala Leu Tyr Pro Pro 610 615 620Pro Pro Pro Glu Asp Lys Ala
Lys Lys Gly Gly Lys Lys Lys Gly Gly625 630 635 640Ser Met Gln Thr
Val Ser Ser Gln Phe Arg Glu Asn Leu His Lys Leu 645 650 655Met Thr
Asn Leu Arg Ser Thr His Pro His Phe Val Arg Cys Leu Ile 660 665
670Pro Asn Glu Ser Lys Thr Pro Gly Leu Met Glu Asn Phe Leu Val Ile
675 680 685His Gln Leu Arg Cys Asn Gly Val Leu Glu Gly Ile Arg Ile
Cys Arg 690 695 700Lys Gly Phe Pro Ser Arg Ile Ile Tyr Ala Asp Phe
Lys Gln Arg Tyr705 710 715 720Lys Val Leu Asn Ala Ser Val Ile Pro
Glu Gly Gln Phe Met Asp Asn 725 730 735Lys Lys Ala Ser Glu Lys Leu
Leu Gly Ser Ile Asp Val Asn His Glu 740 745 750Asp Tyr Lys Phe Gly
His Thr Lys Val Phe Phe Lys Ala Gly Leu Leu 755 760 765Gly Val Leu
Glu Glu Met Arg Asp Glu Lys Leu Ala Ser Leu Val Gly 770 775 780Met
Leu Gln Ala Leu Ser Arg Gly Phe Leu Met Arg Arg Glu Phe Thr785 790
795 800Lys Met Met Glu Arg Arg Glu Ser Ile Tyr Ser Ile Gln Tyr Asn
Ile 805 810 815Arg Ser Phe Met Asn Val Lys Thr Trp Pro Trp Met Lys
Leu Tyr Phe 820 825 830Lys Ile Lys Pro Leu Leu Gln Ser Ala Glu Thr
Glu Lys Glu Leu Ala 835 840 845Asn Met Lys Glu Asn Tyr Glu Lys Met
Lys Thr Asp Leu Ala Lys Ala 850 855 860Leu Ser Thr Lys Lys Gln Met
Glu Glu Lys Leu Val Ser Leu Thr Gln865 870 875 880Glu Lys Asn Asp
Leu Ala Leu Gln Val Ala Ser Glu Gly Glu Ser Leu 885 890 895Asn Asp
Ala Glu Glu Arg Cys Glu Gly Leu Ile Lys Ser Lys Ile Gln 900 905
910Gln Glu Ala Lys Leu Lys Glu Thr Thr Glu Arg Leu Glu Asp Glu Glu
915 920 925Glu Ile Asn Ala Glu Leu Thr Ala Lys Lys Arg Lys Leu Glu
Asp Glu 930 935 940Cys Ser Glu Leu Lys Lys Asp Ile Asp Asp Leu Glu
Leu Thr Leu Ala945 950 955 960Lys Val Glu Lys Glu Lys His Ala Thr
Glu Asn Lys Val Lys Asn Leu 965 970 975Thr Glu Glu Met Ala Ser Met
Asp Glu Ser Val Ala Lys Leu Thr Lys 980 985 990Glu Lys Lys Ala Leu
Gln Glu Ala His Gln Gln Thr Leu Asp Asp Leu 995 1000 1005Gln Ala
Glu Glu Asp Lys Val Asn Thr Leu Thr Lys Ala Lys Thr 1010 1015
1020Lys Leu Glu Gln Gln Val Asp Asp Leu Glu Gly Ser Leu Glu Gln
1025 1030 1035Glu Lys Lys Leu Arg Met Asp Leu Glu Arg Ser Lys Arg
Lys Leu 1040 1045 1050Glu Gly Asp Leu Lys Leu Ala Gln Glu Ser Ile
Met Asp Leu Glu 1055 1060 1065Asn Asp Lys Gln Gln Ala Asp Glu Lys
Ile Lys Lys Lys Glu Phe 1070 1075 1080Glu Thr Thr Gln Leu Leu Ser
Lys Val Glu Asp Glu Gln Ser Leu 1085 1090 1095Gly Ala Gln Leu Gln
Lys Lys Ile Lys Glu Leu Gln Ala Arg Ile 1100 1105 1110Glu Glu Leu
Glu Glu Glu Ile Glu Ala Glu Arg Ala Ala Arg Ala 1115 1120 1125Lys
Val Glu Lys Gln Arg Ala Asp Leu Ser Arg Glu Leu Glu Glu 1130 1135
1140Ile Ser Glu Arg Leu Glu Glu Ala Gly Gly Ala Thr Ala Ala Gln
1145 1150 1155Ile Glu Met Asn Lys Lys Arg Glu Ala Glu Phe Gln Lys
Leu Arg 1160 1165 1170Arg Asp Leu Glu Glu Ser Thr Leu Gln His Glu
Ala Thr Ala Ala 1175 1180 1185Ala Leu Arg Lys Lys Gln Ala Asp Ser
Val Ala Glu Leu Gly Glu 1190 1195 1200Gln Ile Asp Asn Leu Gln Arg
Val Lys Gln Lys Leu Glu Lys Glu 1205 1210 1215Lys Ser Glu Tyr Lys
Met Glu Ile Asp Asp Leu Ser Ser Asn Met 1220 1225 1230Glu Ala Val
Ala Lys Ala Lys Gly Asn Leu Glu Lys Met Cys Arg 1235 1240 1245Thr
Leu Glu Asp Gln Leu Ser Glu Leu Lys Thr Lys Asn Asp Glu 1250 1255
1260Asn Val Arg Gln Val Asn Asp Ile Ser Gly Gln Arg Ala Arg Leu
1265 1270 1275Leu Thr Glu Asn Gly Glu Phe Gly Arg Gln Leu Glu Glu
Lys Glu 1280 1285 1290Ala Leu Val Ser Gln Leu Thr Arg Gly Lys Gln
Ala Phe Thr Gln 1295 1300 1305Gln Val Glu Glu Leu Lys Arg Ala Ile
Glu Glu Glu Val Lys Ala 1310 1315 1320Lys Asn Ala Leu Ala His Gly
Val Gln Ser Ala Arg His Asp Cys 1325 1330 1335Asp Leu Leu Arg Glu
Gln Phe Glu Glu Glu Gln Glu Ala Lys Ala 1340 1345 1350Glu Leu Gln
Arg Gly Met Ser Lys Ala Asn Ser Glu Val Ala Gln 1355 1360 1365Trp
Arg Thr Lys Tyr Glu Thr Asp Ala Ile Gln Arg Thr Glu Glu 1370 1375
1380Leu Glu Glu Ala Lys Lys Lys Leu Ala Gln Arg Leu Gln Asp Ala
1385 1390 1395Glu Glu Thr Ile Glu Ala Thr Asn Ser Lys Cys Ala Ser
Leu Glu 1400 1405 1410Lys Thr Lys Gln Arg Leu Gln Gly Glu Val Glu
Asp Leu Met Ile 1415 1420 1425Asp Val Glu Arg Ala Asn Ala Leu Ala
Ala Asn Leu Asp Lys Lys 1430 1435 1440Gln Arg Asn Phe Asp Lys Val
Leu Ala Glu Trp Lys Gln Lys Tyr 1445 1450 1455Glu Glu Gly Gln Ala
Glu Leu Glu Gly Ala Gln Lys Glu Ala Arg 1460 1465 1470Ser Met Ser
Thr Glu Leu Phe Lys Met Lys Asn Ser Tyr Glu Glu 1475 1480 1485Ala
Leu Asp His Leu Glu Thr Leu Lys Arg Glu Asn Lys Asn Leu 1490 1495
1500Gln Gln Glu Ile Ser Asp Leu Thr Glu Gln Ile Gly Glu Thr Gly
1505 1510 1515Lys Ser Ile His Glu Leu Glu Lys Ala Lys Lys Thr Val
Glu Thr 1520 1525 1530Glu Lys Ser Glu Ile Gln Thr Ala Leu Glu Glu
Ala Glu Gly Thr 1535 1540 1545Leu Glu His Glu Glu Ser Lys Ile Leu
Arg Val Gln Leu Glu Leu 1550 1555 1560Asn Gln Ile Lys Gly Glu Val
Asp Arg Lys Ile Ala Glu Lys Asp 1565 1570 1575Glu Glu Met Glu Gln
Ile Lys Arg Asn Ser Gln Arg Met Val Asp 1580 1585 1590Ser Met Gln
Ser Thr Leu Asp Ser Glu Val Arg Ser Arg Asn Asp 1595 1600 1605Ala
Leu Arg Val Lys Lys Lys Met Glu Gly Asp Leu Asn Glu Met 1610 1615
1620Glu Ile Gln Leu Ser His Ser Asn Arg Gln Ala Ala Glu Ala Gln
1625 1630 1635Lys Gln Leu Arg Asn Val Gln Gly Gln Leu Lys Asp Ala
Gln Leu 1640 1645 1650His Leu Asp Asp Ala Val Arg Val Ala Glu Asp
Met Lys Glu Gln 1655 1660 1665Ala Ala Met Val Glu Arg Arg Asn Gly
Leu Met Val Ala Glu Ile 1670 1675 1680Glu Glu Leu Arg Val Ala Leu
Glu Gln Thr Glu Arg Gly Arg Lys 1685 1690 1695Val Ala Glu Thr Glu
Leu Val Asp Ala Ser Glu Arg Val Gly Leu 1700 1705 1710Leu His Ser
Gln Asn Thr Ser Leu Leu Asn Thr Lys Lys Lys Leu 1715 1720 1725Glu
Thr Asp Leu Val Gln Val Gln Gly Glu Val Asp Asp Ile Val 1730 1735
1740Gln Glu Ala Arg Asn Ala Glu Glu Lys Ala Lys Lys Ala Ile Thr
1745 1750 1755Asp Ala Ala Met Met Ala Glu Glu Leu Lys Lys Glu Gln
Asp Thr 1760 1765 1770Ser Ser His Leu Glu Arg Met Lys Lys Asn Leu
Glu Ile Thr Val 1775 1780 1785Lys Asp Leu Gln His Arg Leu Asp Glu
Ala Glu Asn Leu Ala Met 1790 1795 1800Lys Gly Gly Lys Lys Gln Leu
Gln Lys Leu Glu Ser Arg Val Arg 1805 1810 1815Glu Leu Glu Thr Glu
Val Glu Ala Glu Gln Arg Arg Gly Val Asp 1820 1825 1830Ala Val Lys
Gly Val Arg Lys Tyr Glu Arg Arg Val Lys Glu Leu 1835 1840 1845Thr
Tyr Gln Thr Glu Glu Asp Lys Lys Asn Val Asn Arg Leu Gln 1850 1855
1860Asp Leu Val Asp Lys Leu Gln Met Lys Val Lys Ala Tyr Lys Arg
1865 1870 1875His Ala Glu Glu Ala Glu Glu Ala Ala Asn Gln His Met
Ser Lys 1880 1885 1890Phe Arg Lys Val Gln His Glu Leu Glu Glu Ala
Glu Glu Arg Ala 1895 1900 1905Asp Ile Ala Glu Thr Gln Val Asn Lys
Leu Arg Ala Lys Thr Arg 1910 1915 1920Asp Ser Gly Lys Gly Lys Glu
Val Ala Glu 1925 1930717PRTArtificial Sequencemyosin heavy chain,
fast skeletal muscle-like fragment 71Ala Ala Ile Tyr Leu Arg Lys1
57210PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 72Ala Asp Ile Glu Thr Tyr Leu Leu Glu Lys1 5
107319PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 73Ala Leu Gln Glu Ala His Gln Gln Thr Leu Asp
Asp Leu Gln Ala Glu1 5 10 15Glu Asp Lys7411PRTArtificial
Sequencemyosin heavy chain, fast skeletal muscle-like fragment
74Asp Ile Asp Asp Leu Glu Leu Thr Leu Ala Lys1 5
107511PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 75Glu Ala Leu Val Ser Gln Leu Thr Arg Gly Lys1
5 107610PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 76Glu Phe Glu Thr Thr Gln Leu Leu Ser Lys1 5
107710PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 77Glu Leu Thr Tyr Gln Thr Glu Glu Asp Lys1 5
107815PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 78Glu Asn Gln Ser Val Leu Ile Thr Gly Glu Ser
Gly Ala Gly Lys1 5 10 157919PRTArtificial Sequencemyosin heavy
chain, fast skeletal muscle-like fragment 79Glu Thr Thr Glu Arg Leu
Glu Asp Glu Glu Glu Ile Asn Ala Glu Leu1 5 10 15Thr Ala
Lys8010PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 80Phe Ile Arg Ile His Phe Gln Ala Gly Lys1 5
108112PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 81Gly Phe Pro Ser Arg Ile Ile Tyr Ala Asp Phe
Lys1 5 108210PRTArtificial Sequencemyosin heavy chain, fast
skeletal muscle-like fragment 82Gly Leu Val Thr Ala Arg Ala Asp Gly
Lys1 5 108316PRTArtificial Sequencemyosin heavy chain, fast
skeletal muscle-like fragment 83Gly Gln Thr Val Ala Gln Val Asn Asn
Ser Val Ser Ala Leu Ala Lys1 5 10 158412PRTArtificial
Sequencemyosin heavy chain, fast skeletal muscle-like fragment
84Ile Lys Pro Leu Leu Gln Ser Ala Glu Thr Glu Lys1 5
108514PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 85Ile Leu Ala Ala Leu Tyr Pro Pro Pro Pro Pro
Glu Asp Lys1 5 108612PRTArtificial Sequencemyosin heavy chain, fast
skeletal muscle-like fragment 86Ile Leu Arg Val Gln Leu Glu Leu Asn
Gln Ile Lys1 5 108718PRTArtificial Sequencemyosin heavy chain, fast
skeletal muscle-like fragment 87Leu Ala Gln Arg Leu Gln Asp Ala Glu
Glu Thr Ile Glu Ala Thr Asn1 5 10 15Ser Lys8816PRTArtificial
Sequencemyosin heavy chain, fast skeletal muscle-like fragment
88Leu Glu Gln Gln Val Asp Asp Leu Glu Gly Ser Leu Glu Gln Glu Lys1
5 10 158924PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 89Leu Glu Ser Arg Val Arg Glu Leu Glu Thr Glu
Val Glu Ala Glu Gln1 5 10 15Arg Arg Gly Val Asp Ala Val Lys
209013PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 90Leu Leu Gly Ser Ile Asp Val Asn His Glu Asp
Tyr Lys1 5 109121PRTArtificial Sequencemyosin heavy chain, fast
skeletal muscle-like fragment 91Leu Arg Arg Asp Leu Glu Glu Ser Thr
Leu Gln His Glu Ala Thr Ala1 5 10 15Ala Ala Leu Arg Lys
209212PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 92Leu Thr Gly Ala Val Val His His Gly Asn Leu
Lys1 5 109332PRTArtificial Sequencemyosin heavy chain, fast
skeletal muscle-like fragment 93Asn Asp Glu Asn Val Arg Gln Val Asn
Asp Ile Ser Gly Gln Arg Ala1 5 10 15Arg Leu Leu Thr Glu Asn Gly Glu
Phe Gly Arg Gln Leu Glu Glu Lys 20 25 30947PRTArtificial
Sequencemyosin heavy chain, fast skeletal muscle-like fragment
94Asn Leu Glu Ile Thr Val Lys1 59518PRTArtificial Sequencemyosin
heavy chain, fast skeletal muscle-like fragment 95Asn Leu Gln Gln
Glu Ile Ser Asp Leu Thr Glu Gln Ile Gly Glu Thr1 5 10 15Gly
Lys9614PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 96Asn Ser Tyr Glu Glu Ala Leu Asp His Leu Glu
Thr Leu Lys1 5 109719PRTArtificial Sequencemyosin heavy chain, fast
skeletal muscle-like fragment 97Gln Ala Asp Ser Val Ala Glu Leu Gly
Glu Gln Ile Asp Asn Leu Gln1 5 10 15Arg Val Lys9811PRTArtificial
Sequencemyosin heavy chain, fast skeletal muscle-like fragment
98Gln Ala Phe Thr Gln Gln Val Glu Glu Leu Lys1 5
109916PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 99Gln Arg Glu Glu Gln Ala Glu Pro Asp Gly Thr
Glu Val Ala Asp Lys1 5 10 151008PRTArtificial Sequencemyosin heavy
chain, fast skeletal muscle-like fragment 100Arg Ala Ile Glu Glu
Glu Val Lys1 510120PRTArtificial Sequencemyosin heavy chain, fast
skeletal muscle-like fragment 101Ser Glu Ile Gln Thr Ala Leu Glu
Glu Ala Glu Gly Thr Leu Glu His1 5 10 15Glu Glu Ser Lys
2010212PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 102Thr Val Arg Asn Asp Asn Ser Ser Arg Phe Gly
Lys1 5 1010327PRTArtificial Sequencemyosin heavy chain, fast
skeletal muscle-like fragment 103Val Ala Glu Thr Glu Leu Val Asp
Ala Ser Glu Arg Val Gly Leu Leu1 5 10 15His Ser Gln Asn Thr Ser Leu
Leu Asn Thr Lys 20 2510413PRTArtificial Sequencemyosin heavy chain,
fast skeletal muscle-like fragment 104Val Glu Asp Glu Gln Ser Leu
Gly Ala Gln Leu Gln Lys1 5 101058PRTArtificial Sequencemyosin heavy
chain, fast skeletal muscle-like fragment 105Val Gly Asn Glu Tyr
Val Thr Lys1 510621PRTArtificial Sequencemyosin heavy chain, fast
skeletal muscle-like fragment 106Val Gln His Glu Leu Glu Glu Ala
Glu Glu Arg Ala Asp Ile Ala Glu1 5 10 15Thr Gln Val Asn Lys
2010713PRTArtificial Sequencemyosin heavy chain, fast skeletal
muscle-like fragment 107Tyr Glu Glu Gly Gln Ala Glu Leu Glu Gly Ala
Gln Lys1 5 1010816PRTArtificial
Sequencemyosin heavy chain, fast skeletal muscle-like fragment
108Tyr Glu Thr Asp Ala Ile Gln Arg Thr Glu Glu Leu Glu Glu Ala Lys1
5 10 151092532DNAArtificial Sequenceglycogen phosphorylase, muscle
form-like 109atgtcaaaac cattgtcaga ccacgataaa aggaagcaga tctccgtgag
aggtcttgct 60ggtgtggaaa atgtcgcaga actgaaggtc gctttcaaca ggcatctcca
ttttacactg 120gtcaaggaca gaaatgtggc aaacaaacgg gattactact
ttgctctcgc caacaccgtg 180cgtgaccact tggtgggcag gtggatcaga
acccagcagt actactatga gaaagatccc 240aaacgtgtgt actacatctc
cctggagttt tatatgggtc gcaccctgca gaacactatg 300gtgaacctgg
ccctggagaa cgcttgtgat gaggccatat accagctggg tctggacatg
360gaggagctgg aggacatgga agaggatgct ggcctgggaa acggtggtct
tggccgtctt 420gccgcatgct tcctggactc aatggcttct ctaggccttg
ctgcctatgg ctatggtatc 480cgctatgagt ttggcatctt caatcagaag
atcgtcaatg gctggcaggt tgaggaggcc 540gatgattggt tgcgttacgg
caacccctgg gagaaggccc gccctgagta catgcgcccc 600gtcaagttct
atggcaggac cgagcacacc ccagatggtg tgaaatgggt tgacactcag
660gtagtgttgg ctctgccata tgacacccct attcccggct acagaaacaa
cattgtcaac 720accatgagac tgtggtctgc taaggcccca tgcgacttca
acctgaaaga cttcaacgtt 780ggtggctaca ttcaggctgt gttggacaga
aacttgtgtg agaacatttc ccgcgtgctg 840taccccaatg ataatttctt
tgagggcaag gagctgcgtc tgaagcagga gtactttgtg 900gtggccgcca
ctctgcagga cgtcgtccgt cgtttcaagg cctctaagtt tggctccaga
960gagattgtcc gcacagactt cgccgagcta ccaaacaaag ttgccatcca
gctgaatgac 1020actcaccctg ccatggctat tcctgagctg atgagggtcc
tggttgatga ggagaagctg 1080ggttgggaca aggcctggga cgtgtgtgtc
cgtacctgtg cctacacaaa ccacaccgtg 1140ctgcctgagg ccctggagcg
ctggcccatt gacctgttcc atcacctgct gccacgtcac 1200ctggagatca
tctacgagat caaccgtcgc ttcatggagt atgttgcctc gaagttccct
1260ggagacaacg accgtctgcg tcgcatgtcc ctgattgagg agggaggatg
caagaaagtc 1320aacatggctc atctgtgtat cgttggttcc catgctgtca
acggcgtggc ccgcatccac 1380tctgagatcc tcatcgccac tctgttcaag
gacttctatg agttggaccc acacaagttt 1440cagaacaaga ccaatgggat
caccccccgt cgctggctgg ttatgtgcaa ccccggcttg 1500gcggaggtca
tcgcagagaa aattggagag gagtttatcc gtgaccttga ccagcttaag
1560cgactgttga agttcgttaa tgatgatgct ttcatccgtg acatcgccaa
agtcaagcag 1620gagaacaagc tgaagttcgc tgtgcacctg gaagagcact
acaaggtcaa gatcaacccc 1680cagtccatgt ttgacttcca agtcaaaaga
atccacgagt acaaaagaca gctgctcaac 1740tgtctgcaca tgatcaccta
ctacaaccgt atcaagaagg agcccaacaa gcactggacc 1800ccaagaacca
tcatggtcgg aggaaaggct gcaccaggct accacacagc caagatgatc
1860atccgtctca tcacagctat cggtgaggtt gtcaaccacg accccgttgt
cggcgaccgc 1920ctcaaagtta tcttcctgga gaactacaga gtcaccctgg
ctgagaaagc catcccctct 1980gccgacctgt ctgagcagat ctctacagct
ggcaccgagg cctctggcac tggtaacatg 2040aagttcatgc tgaacggtgc
tcttaccatt ggcaccatgg atggagccaa tgtggagatg 2100gccgaggagg
ccggagagga aaacttcttc atcttcggta tgagagtgga ggatgtggac
2160gcaatggacg ccggcaaagg ataccacgcc tctgagtact acaaccgtat
tcccgagctg 2220aagcaggcca tggaccagat tgctggcggc ttcttcagtc
ccaagcagcc tgacctcttc 2280aaggaacttg tggacctgct gatgcaccat
gacaggttca aggtgtttgc tgactacgaa 2340gcctacatca aatgccagga
caaggtcaac caactgtaca agaatcccaa ggaatggacc 2400aagatggtga
tccataacat tgcgggctgt ggtaaattct ccagcgaccg caccattgcc
2460cagtacgccc gagagatctg gggcatggag cccagcctgg agaagatccc
tgcccccgat 2520gagaaactca aa 2532110844PRTArtificial
Sequenceglycogen phosphorylase, muscle form-like 110Met Ser Lys Pro
Leu Ser Asp His Asp Lys Arg Lys Gln Ile Ser Val1 5 10 15Arg Gly Leu
Ala Gly Val Glu Asn Val Ala Glu Leu Lys Val Ala Phe 20 25 30Asn Arg
His Leu His Phe Thr Leu Val Lys Asp Arg Asn Val Ala Asn 35 40 45Lys
Arg Asp Tyr Tyr Phe Ala Leu Ala Asn Thr Val Arg Asp His Leu 50 55
60Val Gly Arg Trp Ile Arg Thr Gln Gln Tyr Tyr Tyr Glu Lys Asp Pro65
70 75 80Lys Arg Val Tyr Tyr Ile Ser Leu Glu Phe Tyr Met Gly Arg Thr
Leu 85 90 95Gln Asn Thr Met Val Asn Leu Ala Leu Glu Asn Ala Cys Asp
Glu Ala 100 105 110Ile Tyr Gln Leu Gly Leu Asp Met Glu Glu Leu Glu
Asp Met Glu Glu 115 120 125Asp Ala Gly Leu Gly Asn Gly Gly Leu Gly
Arg Leu Ala Ala Cys Phe 130 135 140Leu Asp Ser Met Ala Ser Leu Gly
Leu Ala Ala Tyr Gly Tyr Gly Ile145 150 155 160Arg Tyr Glu Phe Gly
Ile Phe Asn Gln Lys Ile Val Asn Gly Trp Gln 165 170 175Val Glu Glu
Ala Asp Asp Trp Leu Arg Tyr Gly Asn Pro Trp Glu Lys 180 185 190Ala
Arg Pro Glu Tyr Met Arg Pro Val Lys Phe Tyr Gly Arg Thr Glu 195 200
205His Thr Pro Asp Gly Val Lys Trp Val Asp Thr Gln Val Val Leu Ala
210 215 220Leu Pro Tyr Asp Thr Pro Ile Pro Gly Tyr Arg Asn Asn Ile
Val Asn225 230 235 240Thr Met Arg Leu Trp Ser Ala Lys Ala Pro Cys
Asp Phe Asn Leu Lys 245 250 255Asp Phe Asn Val Gly Gly Tyr Ile Gln
Ala Val Leu Asp Arg Asn Leu 260 265 270Cys Glu Asn Ile Ser Arg Val
Leu Tyr Pro Asn Asp Asn Phe Phe Glu 275 280 285Gly Lys Glu Leu Arg
Leu Lys Gln Glu Tyr Phe Val Val Ala Ala Thr 290 295 300Leu Gln Asp
Val Val Arg Arg Phe Lys Ala Ser Lys Phe Gly Ser Arg305 310 315
320Glu Ile Val Arg Thr Asp Phe Ala Glu Leu Pro Asn Lys Val Ala Ile
325 330 335Gln Leu Asn Asp Thr His Pro Ala Met Ala Ile Pro Glu Leu
Met Arg 340 345 350Val Leu Val Asp Glu Glu Lys Leu Gly Trp Asp Lys
Ala Trp Asp Val 355 360 365Cys Val Arg Thr Cys Ala Tyr Thr Asn His
Thr Val Leu Pro Glu Ala 370 375 380Leu Glu Arg Trp Pro Ile Asp Leu
Phe His His Leu Leu Pro Arg His385 390 395 400Leu Glu Ile Ile Tyr
Glu Ile Asn Arg Arg Phe Met Glu Tyr Val Ala 405 410 415Ser Lys Phe
Pro Gly Asp Asn Asp Arg Leu Arg Arg Met Ser Leu Ile 420 425 430Glu
Glu Gly Gly Cys Lys Lys Val Asn Met Ala His Leu Cys Ile Val 435 440
445Gly Ser His Ala Val Asn Gly Val Ala Arg Ile His Ser Glu Ile Leu
450 455 460Ile Ala Thr Leu Phe Lys Asp Phe Tyr Glu Leu Asp Pro His
Lys Phe465 470 475 480Gln Asn Lys Thr Asn Gly Ile Thr Pro Arg Arg
Trp Leu Val Met Cys 485 490 495Asn Pro Gly Leu Ala Glu Val Ile Ala
Glu Lys Ile Gly Glu Glu Phe 500 505 510Ile Arg Asp Leu Asp Gln Leu
Lys Arg Leu Leu Lys Phe Val Asn Asp 515 520 525Asp Ala Phe Ile Arg
Asp Ile Ala Lys Val Lys Gln Glu Asn Lys Leu 530 535 540Lys Phe Ala
Val His Leu Glu Glu His Tyr Lys Val Lys Ile Asn Pro545 550 555
560Gln Ser Met Phe Asp Phe Gln Val Lys Arg Ile His Glu Tyr Lys Arg
565 570 575Gln Leu Leu Asn Cys Leu His Met Ile Thr Tyr Tyr Asn Arg
Ile Lys 580 585 590Lys Glu Pro Asn Lys His Trp Thr Pro Arg Thr Ile
Met Val Gly Gly 595 600 605Lys Ala Ala Pro Gly Tyr His Thr Ala Lys
Met Ile Ile Arg Leu Ile 610 615 620Thr Ala Ile Gly Glu Val Val Asn
His Asp Pro Val Val Gly Asp Arg625 630 635 640Leu Lys Val Ile Phe
Leu Glu Asn Tyr Arg Val Thr Leu Ala Glu Lys 645 650 655Ala Ile Pro
Ser Ala Asp Leu Ser Glu Gln Ile Ser Thr Ala Gly Thr 660 665 670Glu
Ala Ser Gly Thr Gly Asn Met Lys Phe Met Leu Asn Gly Ala Leu 675 680
685Thr Ile Gly Thr Met Asp Gly Ala Asn Val Glu Met Ala Glu Glu Ala
690 695 700Gly Glu Glu Asn Phe Phe Ile Phe Gly Met Arg Val Glu Asp
Val Asp705 710 715 720Ala Met Asp Ala Gly Lys Gly Tyr His Ala Ser
Glu Tyr Tyr Asn Arg 725 730 735Ile Pro Glu Leu Lys Gln Ala Met Asp
Gln Ile Ala Gly Gly Phe Phe 740 745 750Ser Pro Lys Gln Pro Asp Leu
Phe Lys Glu Leu Val Asp Leu Leu Met 755 760 765His His Asp Arg Phe
Lys Val Phe Ala Asp Tyr Glu Ala Tyr Ile Lys 770 775 780Cys Gln Asp
Lys Val Asn Gln Leu Tyr Lys Asn Pro Lys Glu Trp Thr785 790 795
800Lys Met Val Ile His Asn Ile Ala Gly Cys Gly Lys Phe Ser Ser Asp
805 810 815Arg Thr Ile Ala Gln Tyr Ala Arg Glu Ile Trp Gly Met Glu
Pro Ser 820 825 830Leu Glu Lys Ile Pro Ala Pro Asp Glu Lys Leu Lys
835 8401119PRTArtificial Sequenceglycogen phosphorylase, muscle
form-like fragment 111Asp Phe Tyr Glu Leu Asp Pro His Lys1
511210PRTArtificial Sequenceglycogen phosphorylase, muscle
form-like fragment 112Phe Ala Val His Leu Glu Glu His Tyr Lys1 5
1011313PRTArtificial Sequenceglycogen phosphorylase, muscle
form-like fragment 113Phe Tyr Gly Arg Thr Glu His Thr Pro Asp Gly
Val Lys1 5 1011415PRTArtificial Sequenceglycogen phosphorylase,
muscle form-like fragment 114Gly Tyr His Ala Ser Glu Tyr Tyr Asn
Arg Ile Pro Glu Leu Lys1 5 10 1511513PRTArtificial Sequenceglycogen
phosphorylase, muscle form-like fragment 115Ile Gly Glu Glu Phe Ile
Arg Asp Leu Asp Gln Leu Lys1 5 1011614PRTArtificial
Sequenceglycogen phosphorylase, muscle form-like fragment 116Gln
Ala Met Asp Gln Ile Ala Gly Gly Phe Phe Ser Pro Lys1 5
1011717PRTArtificial Sequenceglycogen phosphorylase, muscle
form-like fragment 117Gln Ile Ser Val Arg Gly Leu Ala Gly Val Glu
Asn Val Ala Glu Leu1 5 10 15Lys11810PRTArtificial Sequenceglycogen
phosphorylase, muscle form-like fragment 118Val Phe Ala Asp Tyr Glu
Ala Tyr Ile Lys1 5 1011914PRTArtificial Sequenceglycogen
phosphorylase, muscle form-like fragment 119Val Ile Phe Leu Glu Asn
Tyr Arg Val Thr Leu Ala Glu Lys1 5 101203399DNAArtificial
Sequencemyosin-binding protein C, fast-type-like 120atgcctgaag
ccgctgatgc agtgaagccg gagggcgagg agggagaagc accagcagaa 60gccgaagaaa
atactgcaga agatgatgag ccactgcctg aaggagagga gggtgcccat
120cagtcacagg agattacagg actgtttctt cagcaccctg agactacggt
tgccataaca 180ggtacggaca ttgtgtttac tgccaaagtg gactctacta
cactatcaag gaaacccacc 240attaaatggc tgaaggggaa gtggctggac
ctgggcagca aagcaggaaa acacatgcag 300ttcaaagaga cttttgacag
agccacaaag atctacacct gggacatgaa gataatcaag 360gtggtccctg
gtgacgctgg ggcctacagg tgtgaggtca cctccaaaga caagtgtgac
420agcagcgctt tcgacatctc cgtggaggct gtgcaaattg aggaggaaca
tgattctttg 480gccgcattca aaagaacgga tgctggagag gatgaaggca
gtttggattt cagtgctttg 540ctgaaagcta ccaagaagaa gaagaagcca
gtcgtagatg aggagaaggc agacgtgtgg 600gaaatcctga agaacgctca
acccagcgag tatgagaaga ttgcttttga gtacggcatt 660acagacctga
ggggtctgct caagcgactg aagaagatga agactgttga gcccaagcac
720agcgacgctt tcctaaagag aatggagtct gcctactctg tggataaggg
caagaaaatc 780gtactgcagg tggaagtggt tgacccaaat gcccaggtca
aatggttgaa gaacggtcag 840gagataaaat catcagccaa gtacatcata
gaatcagttg gcaacatcag gacgctcacc 900atcaacaagt gtagcctggc
tgacgacgct gcgtacgagt gtgtgattgg ggaagagaag 960tccttcacag
aggtgtttgt caaagagccc ccggtcacca tcaccaagct gctggatgat
1020taccacgtgg tggtgggaga gagagtggag tttgaggtgg aggtgtctgt
ggagggagca 1080cacgttaact ggatgtttga ggaccaagaa ctctccaggg
acacccataa gtaccgcttt 1140aagaaggacg ggttgaagca catgctcatc
atccaagagg ctacgctgga tgacattgga 1200atgtactggt gcttcaccaa
cggcgggcga accaaaggag agctagaagt tgaagagaaa 1260gaactggagg
tgttgcagaa catcgctgac ctgacggtga aggccgagga ccaggccatg
1320ttcaagtgtg aggtgtctga tgagaaggta acagggaaat ggctcaaaga
tggcgtggag 1380gtgctgccca gcagtcgcat caagctgacc cacatcggaa
gaatccatcg gcttaccata 1440gacgacgtca agcccgagga cgctggagac
tataccttta tcccagacgg atatgccctc 1500tcactgtccg ccaaactcaa
cttcctggaa attaagatcg actatgtccc ccgccaagag 1560ccacccaaga
tccatctaga caccaccggc aacatggttt cccagaacac catcattgtg
1620gtggctggca acaagctgcg cctggacgtg gagatctctg gagagcctcc
tcccactgtc 1680gtctgggcaa agggagacac ggcaatcaca gctgtggagg
ggcgtgtgag gactgagagc 1740cggaaggacc tgagctgctt cgtcatagag
ggggcagaga gagaggatga gggcaactac 1800accatcattg tcaccaaccc
tgccggagag gacaaggctc atctgtttgt caagattgtt 1860gatgtgcctg
actgccctga gaatgtaaag tgtacctcgg tgggagaaga ctgtgccacc
1920atggtctggg acccacccaa gttcgacgga ggcgcaccag tcacaggcta
tctcatggag 1980aggaagaaga agggctccac caggtggacg aagctcaact
ttgacgtgta cgagggggtg 2040acgtacgaag ccaagaggat gattgagggc
gtgctctacg agatgagagt gtacgccgtc 2100aatggcattg gcatgtccca
gcccagcctc aattccaaac ccttcatgcc catcgctgca 2160accagtgagc
ctctgggcct caaggtgcat gatgtcacag acaccacatg taccctgaag
2220tggctggccc ctgagaagat cggtgccgga ggcctggacg gatacgttat
tgagtactgc 2280aaggagggag acacggagtg ggtggtggcg aacactgagc
tggtggagag acagaattac 2340acagtccgca acctccccac cgcggagaag
atcaacttca gagtggtggc cgtgaacatt 2400gctggacgca gcccccctac
cggcctgagc cagcccgtca ctatccgcga gatcgtggaa 2460ctgcccaaga
tccgcctccc tcgctacttg agacagaagt acattagaag agtgggcgac
2520aaaatcaacc tgaccatccc cttccagggt aagccacgtc ccaaagtgca
atggtacaag 2580gatggggaag agcttgacac tcgagttgcc agcatacgca
attccgaagt ggactctatc 2640ctgttcatcc gctcggctga gagatcccac
tctgggaagt acgagttggt cctgcagatt 2700gagaacatgg aggccagggc
tattctggaa atcaggatcg tagaggcacc tgggcctccc 2760gaggtagtga
aggtcacaga cgtttggggc ttcaacgctg cgctggagtg gaagccaccg
2820aaggacgacg gcaactgcga gatcaccggt tacacaatac agaaggcgga
caagaagaca 2880aatgaatggt tcactatcta cgagcacaac aggaggacaa
actgcacagc ctcagacctg 2940atcatgggaa atgagtacat gttccgtgtc
ttcagcgaga acctcgttgg aaagagtgag 3000gatttctgcc tcagcaagga
cacagccatc atacctaaga taggattgga gtacaaccca 3060cctccattca
aggagaagga tatgcaaagc gcccccaagt tcatacagcc cctgcttgac
3120aggtctgtgg tggcaggcta cagtaccacc atcagctctg ctgtcaagac
ttgtcctaaa 3180cctaagatca ggtggttgag gaataagatt cccctggacg
agaaccctag gttcctgatg 3240cagaacaacc agggggtttt gaccctgaac
atccgcaagc ccagtcagta tgacgggggc 3300aagttcacct gcaaggctat
caactctttg ggggaggacg tcgtggagtg caccctactc 3360gtacgagctc
tcaaggacaa ggaagatgga gacgagaaa 33991211133PRTArtificial
Sequencemyosin-binding protein C, fast-type-like 121Met Pro Glu Ala
Ala Asp Ala Val Lys Pro Glu Gly Glu Glu Gly Glu1 5 10 15Ala Pro Ala
Glu Ala Glu Glu Asn Thr Ala Glu Asp Asp Glu Pro Leu 20 25 30Pro Glu
Gly Glu Glu Gly Ala His Gln Ser Gln Glu Ile Thr Gly Leu 35 40 45Phe
Leu Gln His Pro Glu Thr Thr Val Ala Ile Thr Gly Thr Asp Ile 50 55
60Val Phe Thr Ala Lys Val Asp Ser Thr Thr Leu Ser Arg Lys Pro Thr65
70 75 80Ile Lys Trp Leu Lys Gly Lys Trp Leu Asp Leu Gly Ser Lys Ala
Gly 85 90 95Lys His Met Gln Phe Lys Glu Thr Phe Asp Arg Ala Thr Lys
Ile Tyr 100 105 110Thr Trp Asp Met Lys Ile Ile Lys Val Val Pro Gly
Asp Ala Gly Ala 115 120 125Tyr Arg Cys Glu Val Thr Ser Lys Asp Lys
Cys Asp Ser Ser Ala Phe 130 135 140Asp Ile Ser Val Glu Ala Val Gln
Ile Glu Glu Glu His Asp Ser Leu145 150 155 160Ala Ala Phe Lys Arg
Thr Asp Ala Gly Glu Asp Glu Gly Ser Leu Asp 165 170 175Phe Ser Ala
Leu Leu Lys Ala Thr Lys Lys Lys Lys Lys Pro Val Val 180 185 190Asp
Glu Glu Lys Ala Asp Val Trp Glu Ile Leu Lys Asn Ala Gln Pro 195 200
205Ser Glu Tyr Glu Lys Ile Ala Phe Glu Tyr Gly Ile Thr Asp Leu Arg
210 215 220Gly Leu Leu Lys Arg Leu Lys Lys Met Lys Thr Val Glu Pro
Lys His225 230 235 240Ser Asp Ala Phe Leu Lys Arg Met Glu Ser Ala
Tyr Ser Val Asp Lys 245 250 255Gly Lys Lys Ile Val Leu Gln Val Glu
Val Val Asp Pro Asn Ala Gln 260 265 270Val Lys Trp Leu Lys Asn Gly
Gln Glu Ile Lys Ser Ser Ala Lys Tyr 275 280 285Ile Ile Glu Ser Val
Gly Asn Ile Arg Thr Leu Thr Ile Asn Lys Cys 290 295 300Ser Leu Ala
Asp Asp Ala Ala Tyr Glu Cys Val Ile Gly Glu Glu Lys305 310 315
320Ser Phe Thr Glu Val Phe Val Lys Glu Pro Pro Val Thr Ile Thr Lys
325 330 335Leu Leu Asp Asp Tyr His Val Val Val Gly Glu Arg Val Glu
Phe Glu 340 345 350Val Glu Val Ser Val Glu Gly Ala His Val Asn
Trp Met Phe Glu Asp 355 360 365Gln Glu Leu Ser Arg Asp Thr His Lys
Tyr Arg Phe Lys Lys Asp Gly 370 375 380Leu Lys His Met Leu Ile Ile
Gln Glu Ala Thr Leu Asp Asp Ile Gly385 390 395 400Met Tyr Trp Cys
Phe Thr Asn Gly Gly Arg Thr Lys Gly Glu Leu Glu 405 410 415Val Glu
Glu Lys Glu Leu Glu Val Leu Gln Asn Ile Ala Asp Leu Thr 420 425
430Val Lys Ala Glu Asp Gln Ala Met Phe Lys Cys Glu Val Ser Asp Glu
435 440 445Lys Val Thr Gly Lys Trp Leu Lys Asp Gly Val Glu Val Leu
Pro Ser 450 455 460Ser Arg Ile Lys Leu Thr His Ile Gly Arg Ile His
Arg Leu Thr Ile465 470 475 480Asp Asp Val Lys Pro Glu Asp Ala Gly
Asp Tyr Thr Phe Ile Pro Asp 485 490 495Gly Tyr Ala Leu Ser Leu Ser
Ala Lys Leu Asn Phe Leu Glu Ile Lys 500 505 510Ile Asp Tyr Val Pro
Arg Gln Glu Pro Pro Lys Ile His Leu Asp Thr 515 520 525Thr Gly Asn
Met Val Ser Gln Asn Thr Ile Ile Val Val Ala Gly Asn 530 535 540Lys
Leu Arg Leu Asp Val Glu Ile Ser Gly Glu Pro Pro Pro Thr Val545 550
555 560Val Trp Ala Lys Gly Asp Thr Ala Ile Thr Ala Val Glu Gly Arg
Val 565 570 575Arg Thr Glu Ser Arg Lys Asp Leu Ser Cys Phe Val Ile
Glu Gly Ala 580 585 590Glu Arg Glu Asp Glu Gly Asn Tyr Thr Ile Ile
Val Thr Asn Pro Ala 595 600 605Gly Glu Asp Lys Ala His Leu Phe Val
Lys Ile Val Asp Val Pro Asp 610 615 620Cys Pro Glu Asn Val Lys Cys
Thr Ser Val Gly Glu Asp Cys Ala Thr625 630 635 640Met Val Trp Asp
Pro Pro Lys Phe Asp Gly Gly Ala Pro Val Thr Gly 645 650 655Tyr Leu
Met Glu Arg Lys Lys Lys Gly Ser Thr Arg Trp Thr Lys Leu 660 665
670Asn Phe Asp Val Tyr Glu Gly Val Thr Tyr Glu Ala Lys Arg Met Ile
675 680 685Glu Gly Val Leu Tyr Glu Met Arg Val Tyr Ala Val Asn Gly
Ile Gly 690 695 700Met Ser Gln Pro Ser Leu Asn Ser Lys Pro Phe Met
Pro Ile Ala Ala705 710 715 720Thr Ser Glu Pro Leu Gly Leu Lys Val
His Asp Val Thr Asp Thr Thr 725 730 735Cys Thr Leu Lys Trp Leu Ala
Pro Glu Lys Ile Gly Ala Gly Gly Leu 740 745 750Asp Gly Tyr Val Ile
Glu Tyr Cys Lys Glu Gly Asp Thr Glu Trp Val 755 760 765Val Ala Asn
Thr Glu Leu Val Glu Arg Gln Asn Tyr Thr Val Arg Asn 770 775 780Leu
Pro Thr Ala Glu Lys Ile Asn Phe Arg Val Val Ala Val Asn Ile785 790
795 800Ala Gly Arg Ser Pro Pro Thr Gly Leu Ser Gln Pro Val Thr Ile
Arg 805 810 815Glu Ile Val Glu Leu Pro Lys Ile Arg Leu Pro Arg Tyr
Leu Arg Gln 820 825 830Lys Tyr Ile Arg Arg Val Gly Asp Lys Ile Asn
Leu Thr Ile Pro Phe 835 840 845Gln Gly Lys Pro Arg Pro Lys Val Gln
Trp Tyr Lys Asp Gly Glu Glu 850 855 860Leu Asp Thr Arg Val Ala Ser
Ile Arg Asn Ser Glu Val Asp Ser Ile865 870 875 880Leu Phe Ile Arg
Ser Ala Glu Arg Ser His Ser Gly Lys Tyr Glu Leu 885 890 895Val Leu
Gln Ile Glu Asn Met Glu Ala Arg Ala Ile Leu Glu Ile Arg 900 905
910Ile Val Glu Ala Pro Gly Pro Pro Glu Val Val Lys Val Thr Asp Val
915 920 925Trp Gly Phe Asn Ala Ala Leu Glu Trp Lys Pro Pro Lys Asp
Asp Gly 930 935 940Asn Cys Glu Ile Thr Gly Tyr Thr Ile Gln Lys Ala
Asp Lys Lys Thr945 950 955 960Asn Glu Trp Phe Thr Ile Tyr Glu His
Asn Arg Arg Thr Asn Cys Thr 965 970 975Ala Ser Asp Leu Ile Met Gly
Asn Glu Tyr Met Phe Arg Val Phe Ser 980 985 990Glu Asn Leu Val Gly
Lys Ser Glu Asp Phe Cys Leu Ser Lys Asp Thr 995 1000 1005Ala Ile
Ile Pro Lys Ile Gly Leu Glu Tyr Asn Pro Pro Pro Phe 1010 1015
1020Lys Glu Lys Asp Met Gln Ser Ala Pro Lys Phe Ile Gln Pro Leu
1025 1030 1035Leu Asp Arg Ser Val Val Ala Gly Tyr Ser Thr Thr Ile
Ser Ser 1040 1045 1050Ala Val Lys Thr Cys Pro Lys Pro Lys Ile Arg
Trp Leu Arg Asn 1055 1060 1065Lys Ile Pro Leu Asp Glu Asn Pro Arg
Phe Leu Met Gln Asn Asn 1070 1075 1080Gln Gly Val Leu Thr Leu Asn
Ile Arg Lys Pro Ser Gln Tyr Asp 1085 1090 1095Gly Gly Lys Phe Thr
Cys Lys Ala Ile Asn Ser Leu Gly Glu Asp 1100 1105 1110Val Val Glu
Cys Thr Leu Leu Val Arg Ala Leu Lys Asp Lys Glu 1115 1120 1125Asp
Gly Asp Glu Lys 11301228PRTArtificial Sequencemyosin-binding
protein C, fast-type-like fragment 122Ala Asp Val Trp Glu Ile Leu
Lys1 512314PRTArtificial Sequencemyosin-binding protein C,
fast-type-like fragment 123Glu Leu Glu Val Leu Gln Asn Ile Ala Asp
Leu Thr Val Lys1 5 101248PRTArtificial Sequencemyosin-binding
protein C, fast-type-like fragment 124Glu Pro Pro Val Thr Ile Thr
Lys1 51258PRTArtificial Sequencemyosin-binding protein C,
fast-type-like fragment 125Gly Glu Leu Glu Val Glu Glu Lys1
51267PRTArtificial Sequencemyosin-binding protein C, fast-type-like
fragment 126His Ser Asp Ala Phe Leu Lys1 512715PRTArtificial
Sequencemyosin-binding protein C, fast-type-like fragment 127Ile
Ala Phe Glu Tyr Gly Ile Thr Asp Leu Arg Gly Leu Leu Lys1 5 10
1512811PRTArtificial Sequencemyosin-binding protein C,
fast-type-like fragment 128Ile Gly Leu Glu Tyr Asn Pro Pro Pro Phe
Lys1 5 1012914PRTArtificial Sequencemyosin-binding protein C,
fast-type-like fragment 129Ile Asn Leu Thr Ile Pro Phe Gln Gly Lys
Pro Arg Pro Lys1 5 1013015PRTArtificial Sequencemyosin-binding
protein C, fast-type-like fragment 130Ile Val Leu Gln Val Glu Val
Val Asp Pro Asn Ala Gln Val Lys1 5 10 1513114PRTArtificial
Sequencemyosin-binding protein C, fast-type-like fragment 131Leu
Asn Phe Asp Val Tyr Glu Gly Val Thr Tyr Glu Ala Lys1 5
101327PRTArtificial Sequencemyosin-binding protein C,
fast-type-like fragment 132Leu Asn Phe Leu Glu Ile Lys1
51339PRTArtificial Sequencemyosin-binding protein C, fast-type-like
fragment 133Asn Ala Gln Pro Ser Glu Tyr Glu Lys1
513418PRTArtificial Sequencemyosin-binding protein C,
fast-type-like fragment 134Arg Thr Asp Ala Gly Glu Asp Glu Gly Ser
Leu Asp Phe Ser Ala Leu1 5 10 15Leu Lys1358PRTArtificial
Sequencemyosin-binding protein C, fast-type-like fragment 135Ser
Phe Thr Glu Val Phe Val Lys1 513613PRTArtificial
Sequencemyosin-binding protein C, fast-type-like fragment 136Val
Asp Ser Thr Thr Leu Ser Arg Lys Pro Thr Ile Lys1 5
101371551DNAArtificial SequenceATP synthase subunit beta,
mitochondrial 137atgttaggag ctgtgggacg ctgctgcacc ggggctctcc
aggcacttaa gcctggggtc 60cagcccctga aggcccttgt aggatcgcca tctgtccttg
gacgcagaga ctactctgca 120cctgctgctg ctgccgcctt cgcccatggc
aggatcgtag cggtcatcgg tgccgtcgtc 180gacgtccagt tcgatgaggg
cctcccaccc atcctcaatg ccctggaagt tgctgggcgt 240gagtccaggc
tagttctgga ggtggcacag catcttggtg agaacactgt gcgtaccatt
300gccatggatg gtactgaagg tcttgtccgt ggacagaagg ttgtggacac
tggcgatccc 360atcagaatcc cagtgggtcc cgagacccta ggcagaatca
tgaatgtcat tggagagccc 420attgatgaga gggggccaat ctccaccaag
cagactgccg ccatccacgc tgaggcccca 480gagttcactg acatgagtgt
ggagcaggag atcctggtaa ctggcatcaa ggtggtagat 540ctgctggctc
cctatgccaa gggaggcaaa atcggtctgt tcggtggtgc tggtgtgggc
600aagactgtgt tgatcatgga gctgatcaac aatgtggcca aggcccatgg
tggttactct 660gtgtttgccg gagtgggaga gcgtacccgc gagggaaatg
acttgtacca cgagatgatt 720gagtcgggtg tcatcaacct gaaggatgac
acctccaagg tggcgctggt gtacggacaa 780atgaacgagc ccccaggcgc
ccgtgcccgt gtggctctga ctggtctgac cgtggcagag 840tatttccgtg
accaggaggg tcaggatgtg ctgctcttca tcgataacat cttccgtttc
900acccaggctg gctccgaggt gtctgccctg ctgggtcgta tcccctccgc
tgtgggttac 960cagcccaccc tggccactga catgggtacc atgcaggaga
gaatcaccac caccaagaag 1020ggttccatca cctctgtgca ggccatctat
gtgccggctg acgatttgac tgatcccgcc 1080cctgccacca ccttcgctca
cttggacgcc accactgtgc tgtcccgtgc aatcgctgag 1140ctgggtatct
accctgctgt ggatcccctg gattccacct cccgtatcat ggaccccaac
1200attgtcggcg ctgagcacta tgatgttgct cgtggcgtgc agaagatcct
ccaggactac 1260aagtccctgc aggatatcat tgccatcctg ggtatggatg
agttgtctga ggaggacaag 1320ctgattgtct ctcgcgcccg caagatccag
cgtttcctgt cccagccctt ccaggtggcg 1380gaggtattca ccggtcatgc
tggcaagctg gtgccactca aggagaccat cagtggcttc 1440cagagcattc
taaatggtga gtacgatgcc ctgcccgagc aggctttcta catggttgga
1500cccatcgagg aggtggttga gaaggctgca cagatggcca aggatctcgc a
1551138517PRTArtificial SequenceATP synthase subunit beta,
mitochondrial 138Met Leu Gly Ala Val Gly Arg Cys Cys Thr Gly Ala
Leu Gln Ala Leu1 5 10 15Lys Pro Gly Val Gln Pro Leu Lys Ala Leu Val
Gly Ser Pro Ser Val 20 25 30Leu Gly Arg Arg Asp Tyr Ser Ala Pro Ala
Ala Ala Ala Ala Phe Ala 35 40 45His Gly Arg Ile Val Ala Val Ile Gly
Ala Val Val Asp Val Gln Phe 50 55 60Asp Glu Gly Leu Pro Pro Ile Leu
Asn Ala Leu Glu Val Ala Gly Arg65 70 75 80Glu Ser Arg Leu Val Leu
Glu Val Ala Gln His Leu Gly Glu Asn Thr 85 90 95Val Arg Thr Ile Ala
Met Asp Gly Thr Glu Gly Leu Val Arg Gly Gln 100 105 110Lys Val Val
Asp Thr Gly Asp Pro Ile Arg Ile Pro Val Gly Pro Glu 115 120 125Thr
Leu Gly Arg Ile Met Asn Val Ile Gly Glu Pro Ile Asp Glu Arg 130 135
140Gly Pro Ile Ser Thr Lys Gln Thr Ala Ala Ile His Ala Glu Ala
Pro145 150 155 160Glu Phe Thr Asp Met Ser Val Glu Gln Glu Ile Leu
Val Thr Gly Ile 165 170 175Lys Val Val Asp Leu Leu Ala Pro Tyr Ala
Lys Gly Gly Lys Ile Gly 180 185 190Leu Phe Gly Gly Ala Gly Val Gly
Lys Thr Val Leu Ile Met Glu Leu 195 200 205Ile Asn Asn Val Ala Lys
Ala His Gly Gly Tyr Ser Val Phe Ala Gly 210 215 220Val Gly Glu Arg
Thr Arg Glu Gly Asn Asp Leu Tyr His Glu Met Ile225 230 235 240Glu
Ser Gly Val Ile Asn Leu Lys Asp Asp Thr Ser Lys Val Ala Leu 245 250
255Val Tyr Gly Gln Met Asn Glu Pro Pro Gly Ala Arg Ala Arg Val Ala
260 265 270Leu Thr Gly Leu Thr Val Ala Glu Tyr Phe Arg Asp Gln Glu
Gly Gln 275 280 285Asp Val Leu Leu Phe Ile Asp Asn Ile Phe Arg Phe
Thr Gln Ala Gly 290 295 300Ser Glu Val Ser Ala Leu Leu Gly Arg Ile
Pro Ser Ala Val Gly Tyr305 310 315 320Gln Pro Thr Leu Ala Thr Asp
Met Gly Thr Met Gln Glu Arg Ile Thr 325 330 335Thr Thr Lys Lys Gly
Ser Ile Thr Ser Val Gln Ala Ile Tyr Val Pro 340 345 350Ala Asp Asp
Leu Thr Asp Pro Ala Pro Ala Thr Thr Phe Ala His Leu 355 360 365Asp
Ala Thr Thr Val Leu Ser Arg Ala Ile Ala Glu Leu Gly Ile Tyr 370 375
380Pro Ala Val Asp Pro Leu Asp Ser Thr Ser Arg Ile Met Asp Pro
Asn385 390 395 400Ile Val Gly Ala Glu His Tyr Asp Val Ala Arg Gly
Val Gln Lys Ile 405 410 415Leu Gln Asp Tyr Lys Ser Leu Gln Asp Ile
Ile Ala Ile Leu Gly Met 420 425 430Asp Glu Leu Ser Glu Glu Asp Lys
Leu Ile Val Ser Arg Ala Arg Lys 435 440 445Ile Gln Arg Phe Leu Ser
Gln Pro Phe Gln Val Ala Glu Val Phe Thr 450 455 460Gly His Ala Gly
Lys Leu Val Pro Leu Lys Glu Thr Ile Ser Gly Phe465 470 475 480Gln
Ser Ile Leu Asn Gly Glu Tyr Asp Ala Leu Pro Glu Gln Ala Phe 485 490
495Tyr Met Val Gly Pro Ile Glu Glu Val Val Glu Lys Ala Ala Gln Met
500 505 510Ala Lys Asp Leu Ala 51513911PRTArtificial SequenceATP
synthase subunit beta, mitochondrial fragment 139Ile Gly Leu Phe
Gly Gly Ala Gly Val Gly Lys1 5 1014021PRTArtificial SequenceATP
synthase subunit beta, mitochondrial fragment 140Ile Gln Arg Phe
Leu Ser Gln Pro Phe Gln Val Ala Glu Val Phe Thr1 5 10 15Gly His Ala
Gly Lys 2014113PRTArtificial SequenceATP synthase subunit beta,
mitochondrial fragment 141Thr Val Leu Ile Met Glu Leu Ile Asn Asn
Val Ala Lys1 5 1014210PRTArtificial SequenceATP synthase subunit
beta, mitochondrial fragment 142Val Val Asp Leu Leu Ala Pro Tyr Ala
Lys1 5 10143996DNAArtificial SequenceL-lactate dehydrogenase A
chain-like 143atgactacca aggagaagct gatcacccat gtgttggctg
gtgagcctgt tggctcccgg 60agcaaggtga cagttgttgg cgtcggcatg gttggcatgg
cctccgcagt cagcgtcctg 120ctcaaggacc tgtgcgatga gctgtgcctg
attgacgtga tggaagataa actgaagggt 180gaggtcatgg acctgcagca
tggcagcctc ttctgcaaga ctcacaagat tgtgggcgac 240aaggactaca
gtacgactgc ccactccaag gtggtggtgg tcacagccgg tgctcgtcag
300caagagggtg agagccgtct gaacctggtg cagcgtaacg tcaacatctt
caaattcata 360attccccaga tcgtcaagta cagccccaac gccatcctgc
tggtcgtctc caatcctgtt 420gacatcctaa cctacgtggc ttggaagctg
agtggtttcc cccgtcaccg cgtcatcggt 480tccggcacca acctggactc
tggtcgtttc cgccacctga tgggcgagaa gctacacctt 540cacccatcca
gctgtcacgg ctggatcatt ggagaacacg gagactccag cgtgcccgta
600tggagcggtg tgaatgttgc cggtgtttcc ctgaagggcc tgaacccaga
catgggcaca 660gacgcagaca aggaggactg gaagcacgtc cacaagatgg
tggtcgacgg tgcctatgag 720gtcatcaagc tgaagggtta cacctcctgg
gctatcggca tgtccgtcgc tgacctggtt 780gagagcatcc tgaagaacct
ccacaaagtc caccctgtgt ccaccctggt caagggaatg 840cacggtgtga
aggaggaggt gtttctcagc gtgccctgcg tgctgggaaa cagcggtctg
900accgacgtca tccacatgac tctgaagccc gaggaggaga agcagctgag
caacagtgcc 960gagaccctat ggggcgtaca gaaagagctc accttg
996144332PRTArtificial SequenceL-lactate dehydrogenase A chain-like
144Met Thr Thr Lys Glu Lys Leu Ile Thr His Val Leu Ala Gly Glu Pro1
5 10 15Val Gly Ser Arg Ser Lys Val Thr Val Val Gly Val Gly Met Val
Gly 20 25 30Met Ala Ser Ala Val Ser Val Leu Leu Lys Asp Leu Cys Asp
Glu Leu 35 40 45Cys Leu Ile Asp Val Met Glu Asp Lys Leu Lys Gly Glu
Val Met Asp 50 55 60Leu Gln His Gly Ser Leu Phe Cys Lys Thr His Lys
Ile Val Gly Asp65 70 75 80Lys Asp Tyr Ser Thr Thr Ala His Ser Lys
Val Val Val Val Thr Ala 85 90 95Gly Ala Arg Gln Gln Glu Gly Glu Ser
Arg Leu Asn Leu Val Gln Arg 100 105 110Asn Val Asn Ile Phe Lys Phe
Ile Ile Pro Gln Ile Val Lys Tyr Ser 115 120 125Pro Asn Ala Ile Leu
Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr 130 135 140Tyr Val Ala
Trp Lys Leu Ser Gly Phe Pro Arg His Arg Val Ile Gly145 150 155
160Ser Gly Thr Asn Leu Asp Ser Gly Arg Phe Arg His Leu Met Gly Glu
165 170 175Lys Leu His Leu His Pro Ser Ser Cys His Gly Trp Ile Ile
Gly Glu 180 185 190His Gly Asp Ser Ser Val Pro Val Trp Ser Gly Val
Asn Val Ala Gly 195 200 205Val Ser Leu Lys Gly Leu Asn Pro Asp Met
Gly Thr Asp Ala Asp Lys 210 215 220Glu Asp Trp Lys His Val His Lys
Met Val Val Asp Gly Ala Tyr Glu225 230 235 240Val Ile Lys Leu Lys
Gly Tyr Thr Ser Trp Ala Ile Gly Met Ser Val 245 250 255Ala Asp Leu
Val Glu Ser Ile Leu Lys Asn Leu His Lys Val His Pro 260 265 270Val
Ser Thr Leu Val Lys Gly Met His Gly
Val Lys Glu Glu Val Phe 275 280 285Leu Ser Val Pro Cys Val Leu Gly
Asn Ser Gly Leu Thr Asp Val Ile 290 295 300His Met Thr Leu Lys Pro
Glu Glu Glu Lys Gln Leu Ser Asn Ser Ala305 310 315 320Glu Thr Leu
Trp Gly Val Gln Lys Glu Leu Thr Leu 325 3301458PRTArtificial
SequenceL-lactate dehydrogenase A chain-like fragment 145Phe Ile
Ile Pro Gln Ile Val Lys1 514616PRTArtificial SequenceL-lactate
dehydrogenase A chain-like fragment 146Leu Ile Thr His Val Leu Ala
Gly Glu Pro Val Gly Ser Arg Ser Lys1 5 10 1514714PRTArtificial
SequenceL-lactate dehydrogenase A chain-like fragment 147Thr His
Val Leu Ala Gly Glu Pro Val Gly Ser Arg Ser Lys1 5
101489PRTArtificial SequenceL-lactate dehydrogenase A chain-like
fragment 148Val His Pro Val Ser Thr Leu Val Lys1
514928PRTArtificial SequenceL-lactate dehydrogenase A chain-like
fragment 149Val Val Val Val Thr Ala Gly Ala Arg Gln Gln Glu Gly Glu
Ser Arg1 5 10 15Leu Asn Leu Val Gln Arg Asn Val Asn Ile Phe Lys 20
25
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References